Ernest Truex began acting at age five and toured through Missouri at age nine as "The Child Wonder in Scenes from Shakespeare". His Broadway debut came in 1908 and he performed in several David Belasco plays and portrayed the titled role in the 1915 musical Very Good Eddie.

He made his film debut in 1913, but did not work in film full time for another twenty years. He tended to play "milquetoast" characters and in The Warrior's Husband he played a "nance". In the 1939 The Adventures of Marco Polo, he played Marco Polo's comical assistant, opposite Gary Cooper.

In 1953-1954, Truex starred together with a young Brandon De Wilde in Jamie on ABC. He played aging Grandpa McHummer striking a bond with young Jamie, his recently orphaned grandson.

He also starred in the first season (1958 – 1959) of CBS's The Ann Sothern Show as Jason Macauley, the manager of the swank Bartley House hotel in New York City. Reta Shaw played his wife, Flora. Ann Tyrrell and Jack Mullaney also co-starred. Macauley was succeeded as the hotel chief after twenty-three episodes by Don Porter, playing the role of James Devery, whom Sothern's character, Katy O'Connor, married in the series finale. In 1960, Truex appeared with Harpo Marx in the episode "Silent Panic" of CBS's anthology series The DuPont Show with June Allyson.

In later life, he became known for playing elderly men on television in works such as Justice, Mr. Peepers, and had the main role in the "Kick the Can" episode of Rod Serling's original The Twilight Zone (With his son Barry). In another Twilight Zone episode, "What You Need," he played a traveling peddler who just happened to have exactly what people needed, just before they knew they needed it.