Tim Holt was born Charles John Holt III on February 5, 1919, in Beverly Hills, California, the son of actor Jack Holt and Margaret Woods. During his early years, he accompanied his father on location, even appearing in an early silent film. He was the inspiration for his father's book, Lance and His First Horse.

During the late 1930s George O'Brien had made a number of low budget Westerns for RKO. Holt later recalled, "I believe George O’Brien quit over money so RKO needed another Western star and I was put forward."

Holt was educated at Culver Military Academy in Culver, Indiana, graduating in 1936. One of his classmates was Budd Boetticher who recalled Holt "used to walk around in our suite of rooms there…and he often had on his .38 revolvers and holster. He’d walk up and down the hall in his bathrobe and practice drawing his guns. He’d say, ‘I’m going to be a western star some day’." Immediately after graduation he went to work in the Hollywood film Business.

Instead he made his debut as Anne Shirley's suitor in Stella Dallas (1937) for Sam Goldwyn – the same role that another film star's son, Douglas Fairbanks Jr., had played in the 1925 version. When told he was given the role his father Jack said, "Fine. Let's have one good actor in the family." Wanger then cast him in I Met My Love Again (1938) and used him for a Technicolor Western, Gold is where you find It. In the latter the Los Angeles Times said Holt "confirms the favourable impression he gave" in Stella Dallas.

His contract with Wanger expired. RKO signed Holt to a seven-year contract in December 1938.

He was meant to play the eldest son in Three Sons (1939) with Edward Ellis but he was withdrawn and replaced by william Gargan. He was also meant to star in a Western, Silver City, with Betty Grable, but it was not made.

Holt usually played a Cowboy who had one or two friends, who occasionally sang. From 1940–42 he made 18 Westerns. His first sidekick was Ray Whitley, who was slightly older than Holt, and who would usually sing a song or two in each film. The other sidekick was a character "Whopper" played by Emmett Lynn and then Lee White. 1942 they were replaced by Cliff Edwards as Ike.

Holt's career received a boost in September 1941 when Orson Welles cast him as the lead in his second film, The Magnificent Ambersons (1942). "It was a lucky decision", Welles later said, calling Holt "one of the most interesting actors that's ever been in American movies". The Washington Post thought Holt "gives an excellent account of himself". The New York Times said he "draws out all the meanness in George's character which is precisely what the role demands." (In 1965 Welles wanted to reshoot the ending with those of the original cast still alive, including Holt, but it did not eventuate.)

RKO announced they had purchased two stories for Holt, Five of Spades (which became The Avenging Rider) and Adventures of Salt Valley. He was always announced for There Goes Lona Henry. Holt was going to enter the army, so RKO quickly put him in six more Westerns: Bandit Ranger (1942), Red River Robin Hood (1942), Pirates of the Prairie (1942), Fighting Frontier (1943), Sagebrush Law (1943) and The Avenging Rider (1942).

His most frequent Director was Lesley Selander and his sidekick in more than 25 of these movies was Richard Martin. Martin played Chito Jose Gonzales Bustamente Rafferty, a character created by Writer Jack Wagner for the 1943 film Bombardier and who had appeared in some Robert Mitchum westerns before being put into Tim Holt films. Stempel:

His post-war career began well when 20th Century Fox borrowed him to play Virgil Earp alongside Henry Fonda's Wyatt Earp in the John Ford western My Darling Clementine (1946). For RKO he appeared in a series of Zane Gray adaptations: Thunder Mountain (1947), Under the Tonto Rim (1947) and Wild Horse Mesa (1947). He also made Western Heritage (1947), which was an original screenplay. The budgets were around $100,000 a film, making them among the more expensive B Westerns.

Holt had been to Oklahoma in 1947 with a rodeo, and met the woman who became his final wife. When his movie series ended he decided to move to Oklahoma for good. "I never did like Hollywood that much… there was nothing magical about it for me", he said.

Dore Schary left RKO in 1948 and the new management was not keen to cast Holt in anything other than Westerns: Rustlers (1949), Stagecoach Kid (1949), Masked Raiders (1949), The Mysterious Desperado (1949) and Riders of the Range (1949). The latter lost $50,000.

In September 1950 it was announced Holt would support Montgomery Clift for Director Nicholas Ray in The Flying Leathernecks (1951) but the film was made without either actor.

Instead it was back to Westerns: Pistol Harvest (1951), Hot Lead (1951), Overland Telegraph (1951), Trail Guide (1952), Road Agent (1952), Target (1952), and finally Desert Passage (1952). The latter lost $30,000 so it was decided to end the series once and for all.

He was absent from the screen for five years until he starred in a horror film, The Monster That Challenged the World, in 1957, Holt said it " wasn’t too bad a picture at all.". Over the next 16 years, he appeared in only two more motion pictures as well as an episode of The Virginian. He was going to make a sequel to Treasure of the Sierra Madre with Nick Adams but Adams died.

However he kept busy managing theatres and making personal appearances. He got a degree in animal nutrition from Iowa, worked as a builder, produced rodeos, staged and performed Western music jamborees, and worked as an advertising manager for a radio station from 1962 onwards. Holt later said of this period:

Tim Holt died from bone cancer on February 15, 1973 in Shawnee, Oklahoma, where he had been managing a radio station. He was interred in the Memory Lane Cemetery in Harrah, Oklahoma. The street where he and his wife had lived in Harrah was subsequently renamed Tim Holt Drive in his honor.

In 1991, Tim Holt was inducted posthumously into the Western Performers Hall of Fame at the National Cowboy & Western Heritage Museum in Oklahoma City, Oklahoma. In 1992, the Golden Boot Awards honored Holt for his lifetime contributions to western cinema.

Holt was next borrowed by Warner Bros for the role for which he is probably best remembered — that of Bob Curtin to Humphrey Bogart's Fred C. Dobbs in John Huston's The Treasure of the Sierra Madre (1948), about two friends who team up to prospect for gold, only to have greed tear apart their partnership. Holt's father also appeared in a small part. Tim Holt tended to be overshone in reviews by Bogart and Walter Huston. However the Chicago Daily Tribune said he was very likeable". The Los Angeles Times said Holt "gives a rare and sincere impression of character in his performance."

Following the war, Holt returned to films and went back to RKO. According to his biographer David Rothel, "No more was he the callow, youthful Cowboy with big, silly grin on his face. Now he exuded a steady, serious no-nonsense type of mature Cowboy who was less impulsive, more contemporary, and somewhat ‘world weary." Tom Stempel argues that "While Holt had lost his baby fat during the war, he still had a wonderful grin and cute dimples. He used the mixture of charm and seriousness very well."