Leon Ames was born Harry Wycoff on January 20, 1902, in Portland, Indiana, son of Charles Elmer Wycoff and his wife Cora A. De Moss. Some sources list his original last name as "Wykoff" or "Waycoff", and in his early films, he acted under the name Leon Waycoff. In 1935, Ames explained that he changed his name because Waycoff was often misspelled and mispronounced. Ames was his mother's maiden name.

Ames made his film debut in Quick Millions (1931). During the 1940s, he was under contract to Metro-Goldwyn-Mayer. He played the important role of Mr. Smith in 1944's massive hit and subsequent classic Meet Me in St. Louis.

Ames was one of the founders of the Screen Actors Guild in 1933. He served as its President in 1957.

In 1938, Ames wed Christine Gossett, who gave up acting to raise a family. They were married until his death in 1993.

Ames appeared in a featured role in The Postman Always Rings Twice (1946) as district attorney Kyle Sackett. He also appeared in the Doris Day-Gordon MacRae film On Moonlight Bay, its sequel, By the Light of the Silvery Moon, and Peyton Place (1957). He played the role of Samuel Eaton, Alfred Eaton's (Paul Newman) father, in From the Terrace (1960). He appeared in the 1961 Walt Disney comedy The Absent-Minded Professor as Medfield College President Rufus Daggett and in the 1963 sequel, Son of Flubber. In 1970, he played Secretary of the Navy Frank Knox in the action war film Tora! Tora! Tora! His last film role was in Peggy Sue Got Married (1986), as Kathleen Turner's character's grandfather Barney Alvorg.

Ames made his debut radio broadcast in January 1947 on Grand Central Station.

His television roles included leads in the adaptations of Life With Father (1953–55) and Father of the Bride (1961–62). His presence in the latter program was such that, after the show had been on the air a few months, Ames' role was increased because "'father,' as played by veteran character actor Leon Ames became the dominant figure in the whole show."

He joined the cast of Mister Ed (1963–66) as Wilbur Post's neighbor, retired Colonel Gordon Kirkwood, after the death of actor Larry Keating, who had played Post's original neighbor Roger Addison. Ames also appeared in episodes of the NBC anthology series, The Barbara Stanwyck Show, and on the short-lived CBS legal drama, Storefront Lawyers.

On February 12, 1964, Ames and his wife were held hostage in their home as an intruder demanded $50,000 before he would free them. Ames called his Business partner, who obtained the money from a bank and delivered it to the house as instructed. After inspecting the cash, the kidnapper left Ames in the house, bound with tape, and instructed Mrs. Ames to drive him in the couple's car. He also forced both the Business partner and a guest in the Ames house into the trunk. Eventually, police (who had been alerted by the partner while he was picking up the money) surrounded the car and freed the hostages.

In 1980, after 50 years in show Business, Leon Ames was presented with the Screen Actors Guild Life Achievement Award.

On October 12, 1993, Ames died in Laguna Beach, California, of complications after suffering a stroke. He was 91. He was buried at the Forest Lawn, Hollywood Hills Cemetery in Los Angeles.