Cuoco made her first feature film appearance as the young Karin Carter in the 1995 action thriller Virtuosity. From 2000 to 2001, she appeared on the CBS sitcom Ladies Man and in 2000 she portrayed former The Brady Bunch star Maureen McCormick in the TV film Growing Up Brady as well as starring in the Disney Channel original film Alley Cats Strike. In September 2002, she began a starring role as Bridget Hennessy on the ABC sitcom 8 Simple Rules for Dating My Teenage Daughter (afterwards 8 Simple Rules). Bridget was the oldest child, despite Cuoco being six years younger than Amy Davidson, who played her younger sister.

In September 2007, Cuoco began starring in the CBS sitcom The Big Bang Theory, playing Penny, a Cheesecake Factory employee and aspiring actress who lives across the hall from physicists Dr. Leonard Hofstadter and Dr. Sheldon Cooper. Prior to the 2010–2011 season, she earned US$60,000 an episode for the series, the same as her co-stars. In 2010, the cast negotiated a raise for each actor to make US$200,000 per episode. As of August 2014, Cuoco and her co-stars from The Big Bang Theory Johnny Galecki and Jim Parsons each earned an estimated US$1 million per episode.

Cuoco starred in the Lifetime film To Be Fat like Me, which premiered on January 8, 2007. She played a minor role in the Prison Break episodes “The Message” and “Chicago”, as well as starring in the comedy horror spoof Killer Movie in 2008. She also appeared in the 2010 film The Penthouse. In 2011, she appeared in Allure magazine’s annual “Naked Truth” feature. That same year, she appeared in the films Hop and The Last Ride. She was also chosen to host the Teen Choice Awards in 2011, as well as the People’s Choice Awards in 2012 and 2013. She is a regular on Priceline.com commercials as the daughter of William Shatner’s character.

In 2007, while working on The Big Bang Theory, Cuoco privately dated co-star Johnny Galecki for roughly two years, until December 2009. She later told CBS Watch in September 2010 that they have remained on good terms since ending their relationship.

On September 13, 2010, Cuoco broke her leg in a horse-riding accident and missed the filming of two episodes of The Big Bang Theory.

In October 2011, she became engaged to addiction specialist Josh Resnik, but ended their engagement by March 2012. She became engaged to professional tennis player Ryan Sweeting in September 2013, after three months of dating. They married on December 31, 2013, in Santa Susana, California. On September 25, 2015, it was announced that she and Sweeting had decided to divorce. The divorce was finalized on May 9, 2016.

In December 2014, Cuoco received significant media attention surrounding a remark she made in an interview with Redbook magazine while married to Ryan Sweeting. When asked if she considered herself a feminist, she responded: “Is it bad if I say no? I was never that feminist girl demanding equality, but maybe that’s because I’ve never really faced inequality… I cook for Ryan five nights a week: it makes me feel like a housewife. I love that. I know it sounds old-fashioned, but I like the idea of women taking care of their men”.

In an interview in March 2016, Cuoco said she felt her words were misconstrued, telling Cosmopolitan magazine, “Of course I’m a fucking feminist. Look at me. I bleed feminism: I get equal pay to my male co stars on a big show, I have my own home, I’m as independent as you could possibly be. Maybe I didn’t understand the question, but for me that’s feminism. I’m killing the game, man”.

Cuoco began dating equestrian Karl Cook, son of billionaire Scott Cook, in late 2016. They became engaged on November 30, 2017, Cuoco’s 32nd birthday, and were married on June 30, 2018.

In October 2017, Cuoco founded Yes, Norman Productions, a film and television production company which entered an exclusive multi-year first-look production deal with Warner Bros. Television. The company is named after her dog, Norman. Also in October 2017, The company optioned the rights to The Flight Attendant, a book written by author Chris Bohjalian, which will be developed into a limited series, with Cuoco to star and executive produce.

In October 2018, it was announced that Cuoco would executive produce the DC Universe adult animation series Harley Quinn, and would also voice the title character.

In October 2018, it was announced that Cuoco would executive produce and voice Harley Quinn in the DC Universe adult animation, Harley Quinn. In July 2019, Yes, Norman entered a two-year first look production deal with Warner Bros. Television, and announced that The Flight Attendant had been picked up by WarnerMedia’s upcoming streaming platform, HBO Max. The series is set to premiere on November 26, 2020. In August 2019, it was announced that she would executive produce the upcoming CBS comedy, Pretty, which is written and also executive produced by Lindsey Kraft and Santina Muha.