Pyle was born in Bethune, Colorado on May 11, 1920 to farmer Ben H. Pyle (1895–1988) and his wife Maude (1899–1985); His brother, Willis (1914–2016), was an animator known for his work with Walt Disney Animation Studios and UPA. After graduating from high school, Pyle briefly attended Colorado State University, but dropped out to enter show business. Pyle was a drummer and band member until the United States entered World War II, when he joined the United States Merchant Marine.

Pyle guest-starred 14 times between 1951 and 1953 on the syndicated television series The Range Rider with Jock Mahoney and Dick Jones, appeared as an outlaw in a 1951 episode of the television series The Lone Ranger titled “Backtrail”, episode 71, “The Outcast”, episode 166, “Woman in the White Mask”, and episode 187, “Cross of Santo Domingo”.

In 1953, Pyle appeared as Emil Hatch in episode 46 of The Adventures of Superman entitled “Beware the Wrecker”. He had a part in the 1955 Audie Murphy film To Hell and Back, appeared twice on NBC’s 1955–1956 Western anthology series Frontier (in “Mother of the Brave” and in “The Voyage of Captain Castle”). Pyle appeared twice as an unidentified bank robber in Duncan Renaldo’s syndicated Western series The Cisco Kid. In 1954, he was cast as a henchman of the outlaw Sam Bass in Stories of the Century.

Pyle was cast as Carter in the 1955 episode “Joey’s Father” on Fury. Three years later, he played an arsonist in the episode “The Fire Watchers” of the same series. In 1956, Pyle appeared as Vance Kiley in the episode called “Quicksand” in the TV Western series The Lone Ranger. In 1958, Pyle starred with Judith Evelyn in the episode “Man in the Moon” of the NBC docudrama about the Cold War Behind Closed Doors, hosted by and occasionally starring Bruce Gordon.

In 1955, Pyle married Marilee Carpenter (1924–2010), a production assistant at 20th Century Fox. They had sons David and Tony in 1956 and 1957, respectively. Marilee and Denver divorced in 1970. In 1983, Pyle married Tippie Johnston. That union lasted until his death.

Pyle was twice cast on CBS’s The Public Defender in the role of George Hansen, and three times on the religious anthology series, Crossroads on ABC. He acted the part of a police detective in the 1956 film noir Please Murder Me, starring Raymond Burr.

He appeared as a professor in the syndicated Men into Space series’ 1959 episode “Moonquake”. In an episode of Ripcord, he played a suicidal parachutist. Pyle appeared twice each on the CBS Western series My Friend Flicka and NBC’s The Restless Gun with John Payne. He guest-starred with Grant Withers in the 1959 episode “Tumbleweed Ranger” of Tris Coffin’s syndicated Western series 26 Men, billed as true stories of the Arizona Rangers. He appeared seven times on Richard Boone’s CBS Western Have Gun – Will Travel, his final appearance on the show in 1960 as the character Croft in “The Puppeteer”.

He guest-starred in 1960 in several other Westerns, including Pony Express, The Man from Blackhawk, and Tombstone Territory. He guest-starred in the episode “Trail of the Dead”, the story of five missing prospectors, of Rod Cameron’s modern Western syndicated series State Trooper. He appeared with Sammy Jackson in the episode “Resurrection” of the syndicated American Civil War drama, The Gray Ghost. He was cast as Big Red in the 1959 episode “Woman in the River” of the ABC/Warner Bros. detective series Bourbon Street Beat, starring Andrew Duggan and Richard Long. He made several appearances as Briscoe Darling, Jr., on The Andy Griffith Show.

Pyle was cast in the 1960 episode “Three Wise Men” of ABC’s Stagecoach West as an outlaw who promises to turn himself into the authorities if he can spend Christmas with his family. About this time, Pyle appeared in the segment “Lawyer in Petticoats” of William Bendix’s 1960 NBC Western series Overland Trail, and thereafter in 1961 in “Hand of Vengeance” of the syndicated Western series Two Faces West. Pyle was cast as Jed Corrigan in the 1961 episode “The Tramp” of the NBC family drama series National Velvet.

Pyle guest-starred twice on the CBS series Route 66 with Martin Milner and George Maharis, first in 1961 in the episode “The Newborn” and again in 1962 in “A Long Piece Of Mischief”. He appeared as the father of the doomed family in the dystopian episode “Black Leather Jackets” of The Twilight Zone.

In 1963, Pyle guest-starred on The Dick Van Dyke Show as Uncle George in the episode “Uncle George”.

He appeared in the 1963–1964 season of ABC’s drama about college life Channing. He portrayed the character Brill in the 1964 episode, “Johnny Ride the Pony: One, Two, Three”, of the NBC education drama series, Mr. Novak, starring James Franciscus. Pyle appeared 14 times on Gunsmoke, seven times on Dick Powell’s Zane Grey Theatre, and twice on Frontier Justice, all on CBS. He appeared in seven episodes as Ben Thompson (and twice as other characters) on the ABC Western series The Life and Legend of Wyatt Earp.

Pyle played the frontier character Deadwood Dick in the 1966 episode “The Resurrection of Deadwood Dick” on the syndicated anthology series Death Valley Days.

He also is known for portraying both the suspect and the murder victim on the last original Perry Mason TV episode, “The Case of the Final Fadeout”, in 1966. He was one of 11 actors to hit the Perry Mason trifecta, portraying a victim, a defendant, and the actual murderer (in previous episodes) on the series, which he did in five appearances. Among his other appearances, he played defendant Robert Crane in “The Case of the Deadly Double” in 1958, murderer Tilden Stuart in “The Case of the Jealous Journalist”, and murderer Emery Fillmore in “The Case of the Renegade Refugee” (both in 1961), and murderer Frank Honer in “The Case of the Shifty Shoebox” in 1963.

Pyle portrayed Grandpa Tarleton in all 26 episodes of Tammy in the 1965–1966 season. Pyle portrayed the vengeful Texas Ranger Frank Hamer in the 1967 movie Bonnie and Clyde. He also appeared in an episode of The High Chaparral as a general who had lost his son.

In 1968, he appeared as Titus Purcell, patriarch of a family of homesteaders, in the episode “The Price of Tomatoes” in the sitcom Gomer Pyle, U.S.M.C.. Working for the first time with Jim Nabors playing Gomer Pyle, spun-off from The Andy Griffith Show, he used a screen persona similar to Briscoe Darling, Jr. In 1968, he also directed “The Great Diamond Mines” on Death Valley Days.

Pyle also played in Barnaby Jones in an episode titled “Stalking Horse”(03/18/1976). Pyle played the role of Mad Jack in 36 episodes of the NBC series The Life and Times of Grizzly Adams (1977–1978). He played Buck Webb (Doris Day’s television series father) during the first two seasons of CBS’s The Doris Day Show (1968–1970). He said in 1968 that he based his acting in that role on his father’s personality.

Pyle had a guest-starring role in 1973 on The Streets of San Francisco. In 1975, Walt Disney Productions released a film based on the novel Escape to Witch Mountain. In this film, Tony and Tia were played by Ike Eisenmann and Kim Richards, Lucas Deranian by Donald Pleasence, and the children’s Uncle Bené by Pyle. He appeared as Mayor residing in the town of Purgatory in the first-season episode of Kung Fu, titled “Ancient Warrior”. He also appeared in second-season episode “Crossties” as a doctor. 1985 Season 9 Episode 8 of “The Love Boat”.

Pyle sponsored Uncle Jesse’s Fishing Tournament in Lamar County, Texas. In 10 years of operation, the tournament raised more than $160,000 to support children’s programs there. First established in 1988, the tournament is still going strong and celebrated its 30th anniversary in 2017. It continues to support the children’s charities of Lamar County.

In 1991, the Texas Senate passed a resolution honoring Pyle and his wife for their work with Special Olympics and the Denver Pyle’s Children’s Charities.

In his later life, Pyle played mostly cameo television roles and retired from full-time acting. His last film role was in the 1994 film Maverick. His last known acting role was as Jesse Duke in the 1997 CBS made-for-television movie The Dukes of Hazzard: Reunion!.

Pyle has a star in the Motion Pictures section of the Hollywood Walk of Fame at 7083 Hollywood Boulevard. It was dedicated on December 12, 1997.

Pyle died of lung cancer on Christmas 1997. Memorial services were held January 6, 1998 at First Baptist Church in Waxahachie, Texas. He is buried in an unmarked grave at the Forreston Cemetery in Forreston, Texas. His remains are interred beside those of his second wife’s parents, J.T. Johnston (1914–1993) and Erin Birch Johnston (1913–1989).