Lester A. Burnett (he added the final "e" later in life) was born in Summum, Illinois, on March 18, 1911, and grew up in Ravenwood, Missouri. He began singing as a child and learned to play a wide variety of instruments by ear, yet never learned to read or write music. In his teens, he worked in vaudeville, and starting in 1929, at the state's first commercial radio station, WDZ-AM in Tuscola, Illinois.

His break came in December 1933, when he was hired by Gene Autry to play accordion on National Barn Dance on Chicago's WLS-AM, on which Autry was the major star. As sound films became popular, Hollywood sought musical talent for Western films; and in 1934, Producer Nat Levine cast Autry and Burnette in their film debut (unbilled) as part of a bluegrass band in Mascot Pictures' In Old Santa Fe starring Ken Maynard. Burnette sang and played accordion, and the film included two of his compositions.

He had other small parts until a secondary, but more prominent role in the 1935 serial The Adventures of Rex and Rinty. That same year, Levine gave Autry his first starring role in the 12-part serial The Phantom Empire, with Burnette playing Oscar, a comic-relief role. Mascot was soon absorbed by Republic Pictures, and Republic enjoyed enormous success with its musical Western features starring Autry. In each of the films, Burnette played Autry's comic sidekick, Frog Millhouse, with his trademark floppy black hat and trick voice (imitating a deep, froglike croak). Their association produced 62 feature-length musical Westerns.

In the 1940s, he invented and patented an early home audiovisual system called Cinevision Talkies. Each package contained a 78 rpm record with four of his songs and 15 35 mm slides. The slides were to be projected in order and advanced each time a short tone played on the record during the songs. An inside cover of the record album was white so those with no projector and screen could simply shine a flashlight through the slides and view them on the cover. He also devised more than a dozen clever uses for a Common wire clothes hanger and demonstrated several of them during a TV show guest appearance.

After leaving Republic in June 1944, he became the sidekick to Charles Starrett at Columbia Pictures in the new Durango Kid series. Starrett and Burnette were paired in 56 films, from 1945 to 1952. When Starrett retired, Burnette was still under contract, so Columbia teamed him with Jock Mahoney for a new series of Westerns. A pilot film with Mahoney and Burnette was completed, but never released; Columbia then reassigned Burnette to its Gene Autry series, reuniting Burnette with his former partner.

Burnette enjoyed cooking, and in the 1950s, he opened a restaurant chain called The Checkered Shirt, the first of the A-frame drive-ins. The first location was in Orlando, Florida, and two locations still exist in California (Redding and Escondido), but are no longer owned by the Burnette family.

Gene Autry retired from motion pictures in 1953, and other Cowboy stars had either left the movies or were winding down their screen careers. With the studios no longer interested in making B Westerns, Burnette turned to broadcasting and made guest appearances on many country music radio and TV shows, including Louisiana Hayride, the Grand Ole Opry, and Ranch Party.

In early 1957, when quiz shows were popular, he filmed a pilot for a proposed ABC-TV series to originate from Springfield called Pig 'N Poke, a quiz show with a country theme, although ABC did not buy the show.

In the mid 1960s, he portrayed railway Engineer Charley Pratt on the CBS-TV programs Petticoat Junction (106 episodes) and Green Acres (seven episodes).

Burnette donated his original hat and shirt to the Cowboy Hall of Fame in Oklahoma City, Oklahoma, in 1962. In 1971, he was inducted posthumously into the Nashville Songwriters Hall of Fame.

Just after filming wrapped for the fourth season of Petticoat Junction, Burnette became ill. On February 16, 1967, a month before his 56th birthday, he died in Encino, California, from leukemia and was interred in Forest Lawn Memorial Park in Hollywood Hills, California.

Burnette is mentioned in the Statler Brothers' 1973 country music hit "Whatever Happened to Randolph Scott?" (later the title of a 1994 Scott biography), which reached number 22 on the country chart.

For his contributions to the film industry, Burnette was inducted posthumously into the Hollywood Walk of Fame in 1986 with a motion pictures star located at 6125 Hollywood Boulevard. In 1998, he was inducted into the Western Music Association. On May 5, 2012 he was inducted into the Cowtown Society of Western Music Hall of Fame as a Hero.