Colin Mochrie was born in Kilmarnock, Scotland, the oldest of three children. His father was an airline maintenance executive. He was shy as a child, stating that neighbours would have commented that he “watched way too much television.” In 1964, his family moved to a French neighbourhood just outside Montreal, Quebec, and five years later moved again to Vancouver, British Columbia. Mochrie attended Killarney Secondary School, where he was a self-proclaimed loner who wanted to become a marine biologist. He was persuaded by a friend to try out for a play titled The Death and Life of Sneaky Fitch in which Mochrie played the role of the undertaker. He was hooked when he got his first laugh, which paved the way for a career in entertainment. After graduating from high school as valedictorian, Mochrie attended the Studio 58 theatre school in Vancouver for four years, where he discovered the art of improvisational comedy.

Upon graduation from Studio 58, Mochrie found his first line of work as a member of the Vancouver TheatreSports League. He started working with the group in 1980. Fame was slow to start, as Mochrie “literally had to pull people out of McDonald’s to come see the shows.” Mochrie originally had parts in plays while working for the group, though after a time working for the TheatreSports League became a full-time job for Mochrie. He met fellow improvisor Ryan Stiles during this time. He was visiting a mutual friend in New Zealand when Stiles was doing comedy at Punchlines. After the two met, Stiles and Mochrie began working at TheatreSports together. Though it has been stated that the two met while members of The Second City, the pair were already close friends, according to both Mochrie and Susan Trimbee, the former manager of The Second City Toronto (1985–1988).

Upon finishing his stint with Second City in 1988, Mochrie’s career was quiet for a while as he spent some time with his new wife and child. In 1989, he auditioned for the new British Channel 4 improv show Whose Line Is It Anyway?, but did not make the cut. Mochrie has stated that the audition was a good learning experience because while improv is about setting other people up to be funny, auditions should be about giving yourself the chance to stand out. He moved to Los Angeles the following year, but again auditioned for the British Whose Line, this time making the cut and being asked to fly to London. He appeared on one episode and was again let go. The third time he auditioned, he earned a regular spot on the show. He spent seven years as a regular on the UK version of Whose Line Is It Anyway? and remained a cast member until the show’s end in 1998.

Following Expo ’86, Mochrie ended his tenure with the Vancouver TheatreSports League and moved to Toronto. Once there, Mochrie auditioned for The Second City comedy troupe, where Stiles was working. He began performing with the Second City National Touring Company where he met Debra McGrath, who was the director of the company at the time. The two were married in 1989 and had a child, Kinley, in 1990.

Mochrie remained active elsewhere during his tenure as a Whose Line cast member. In early 1994, he played the role of Mike Brady in a musical version of The Brady Bunch, directed by fellow Second City member Bruce Pirrie. In the production, Mochrie plays the character as caffeine-fuelled, jittery, and neurotic, an exaggeration of the Mike Brady television character, who often had a coffee in his hand on the show. Shortly before his move to the US version of Whose Line in 1998, Mochrie starred in Supertown Challenge as the host of game shows, which the show spoofed. He also appeared in several episodes of the Canadian improvisational comedy series Improv Heaven and Hell.

After the British version of the show ended its run, Mochrie joined the American version of Whose Line? hosted by Drew Carey on ABC. He was brought on alongside Ryan Stiles, who was also a regular member of the UK cast. Mochrie appeared on every episode from its debut in August 1998 to its finale in 2006. He noted his favourite games as “Scenes From a Hat”, where he would have to act out scenes based on suggestions by audience members, and “Whose Line” where he and Stiles would act out a scene and have to add in lines written on pieces of paper. He felt that his weak spots were the musical segments and the “Hoedown” game, which he said was the only time during the show when he felt total fear. Mochrie, who cannot sing, usually spoke his lines instead of singing them.

In an interview, it was revealed that in 1999 Mochrie worked on the Miloš Forman film Man on the Moon, but his scenes were deleted from the final movie. Mochrie was a guest star in three episodes of The Drew Carey Show: “She’s Gotta Have It” (1999), “Drew Live” (1999), and “Drew Live II” (2000). He also appeared on Nickelodeon’s Figure It Out as a celebrity guest panelist; in one segment of the show, he was slimed. He had a one-liner in the “Bad Hare Day” episode of Goosebumps, and he made special guest appearances in several episodes of The Red Green Show.

From 2001 through 2002, Mochrie co-starred in the Canadian comedy series Blackfly for the series’ two seasons. He appeared in This Hour Has 22 Minutes on CBC Television from 2001 through 2003, and on the WB Television Network series Drew Carey’s Green Screen Show in 2004.

In 2003, Mochrie, Leslie Nielsen, Wayne Gretzky, and Roy Halladay appeared in print and television advertisements to encourage people to visit Toronto after the SARS outbreak that struck the city.

In May 2004, he hosted a tongue-in-cheek guide to surviving animal attacks on Animal Planet known as Wild Survival Guide. He has done a commercial supporting Habitat For Humanity. He appeared briefly in a commercial for Buckley’s Cough Syrup, and he was featured in a commercial for New York Fries, manning a steamroller. He appeared on The Tonight Show with Jay Leno as the superhero Overly Sensitive Man (inspired from Whose Line).

By 2004, Mochrie was appearing in so many media spots that a new Canadian television series, Corner Gas, made light of it by having him do a cameo appearance in the tenth episode. In 2005, Mochrie appeared in “Burnt Toast”, a series of eight comedic mini-operas, each depicting a different stage of a romantic relationship in a contemporary setting, produced by Canada’s Rhombus Media. He also appeared in an episode of The Surreal Gourmet. Along with Rosie O’Donnell, Mochrie hosts a video introduction to a tour of the bakery in the Pacific Wharf area of Disney California Adventure Park. In the video, he helps explain how sourdough bread is made. On December 25, 2005, the Canadian Broadcasting Corporation premièred the TV movie The Magical Gathering. Mochrie starred in this, and his daughter Kinley co-starred as Mochrie’s character at a younger age.

According to Mochrie’s agent, Jeff Andrews, during the show’s run Mochrie was better known in Canada as a “commercial king”, performing as characters such the Detergent Crusader for Sunlight detergent. In March 2005, a Nabisco advertising campaign starred Mochrie as the “Snack Fairy”, in which he wears a ballet tutu over ordinary slacks and a shirt. At the end of each commercial, he declares “Snack happy!” and waves his scepter while sporting a smile.

Mochrie starred in Getting Along Famously in 2006. In February 2007, he made a guest appearance as a priest in the seventh episode of Little Mosque on the Prairie, a Canadian television comedy series.

On March 28, 2007, Mochrie and his Whose Line costar Brad Sherwood hosted part of the Press Correspondents’ Dinner with the President. At that event, Sherwood and Mochrie featured Deputy White House Chief of Staff Karl Rove rapping. Rove’s only line was “MC Rove”. On August 29, 2007, it was announced that Mochrie would host the Canadian version of the game show Are You Smarter Than A 5th Grader?. The first of five episodes aired on October 25, 2007. As a result, Mochrie became the fifth member of the American Whose Line? cast to become a game show host, after colleagues Brad Sherwood (The Dating Game and The Big Moment), Greg Proops (VS., Head Games and Rendez-View), Wayne Brady (Don’t Forget the Lyrics! and Let’s Make A Deal), and Drew Carey (Power of 10 and The Price Is Right).

In 2010, he acted in the Canadian television sitcom She’s the Mayor, which debuted in 2011. On July 19, 2010, Mochrie starred as the divorce lawyer working on the case of Spinner and Emma in Degrassi Takes Manhattan.

In 2011, Mochrie appeared as a regular cast member on Drew Carey’s Improv-A-Ganza on GSN. In 2012, Mochrie starred in the ABC improv comedy series Trust Us with Your Life.

Mochrie and Whose Line co-star Brad Sherwood have intermittently toured North America as a two-man stage show since 2002. Initially called “An Evening with Colin and Brad”, they played primarily in small theatre venues. A DVD of their performances, “Colin & Brad: Two Man Group” was released on March 8, 2011.

Mochrie has been nominated for five Canadian Comedy Awards and has won two. He has also won a Gemini Award and a Writers Guild of Canada award for This Hour Has 22 Minutes. In 2013, Mochrie was awarded Canadian Comedy Person of the Year at the Canadian Comedy Awards.

In 2017 Mochrie made a cameo appearance complete with his trademark dry humour, as Ralph Fellows a hotel detective in an episode (11/3) of the Canadian detective TV series Murdoch Mysteries.

Mochrie lives with his wife, Canadian actress Debra McGrath, in Toronto. The two have been married since January 8, 1989 and together, they have a daughter, Kinley Mochrie (b. September 3, 1990, in Toronto, Ontario, Canada) who was originally male (originally named Luke Mochrie) who later transitioned. In 2017, with her permission, Mochrie revealed on Twitter that Kinley is transgender.

As of 2018, Mochrie and Sherwood have continued their performances, currently billed as the “Scared Scriptless Tour”, and playing in larger venues such as the Sydney Opera House, and Royal Albert Hall in London.