His radio career began in 1931, and during the 1930s and 1940s, Tremayne was often heard in more than one show per week. Replacing Don Ameche, he starred in The First Nighter Program from 1936 to 1942. He starred in The Adventures of the Thin Man and The Romance of Helen Trent during the 1940s. He also starred in the title role in The Falcon, and played detective Pat Abbott in The Abbott Mysteries in 1946–47. Tremayne was once named one of the three most distinctive voices on American radio. The other two were Bing Crosby and President Franklin D. Roosevelt.

Tremayne was married four times. He did a morning talk show, The Tremaynes, with his second wife, Alice Reinhart, whom he married December 11, 1945. When Tremayne died in 2003, he was married to his fourth wife, Joan.

Tremayne portrayed Billy Herbert in the television version of One Man's Family (1949-1955) and Inspector Richard Queen in The Further Adventures of Ellery Queen on NBC (1958-1959).

In 1962 Tremayne portrayed the part of C.J. Hasler, a known thief in The Andy Griffith Show episode entitled, "Andy and Barney in the Big City" aired on 26 March 1962. In that show, he played the part of a cunning opportunist who happens onto off-duty Barney Fife who himself believes that he is stalking a jewel thief (Allan Melvin) who is in fact the house detective of the hotel where the story takes place.

In 1963 Tremayne appeared in the Perry Mason episode, "The Case of Constant Doyle", along with special guest attorney Bette Davis. He appeared in seven other episodes as various characters, such as Deputy District Attorney Stewart Linn in the 1960 episode, "The Case of the Madcap Modiste". In 1961 he played the title role of murder victim Willard Nesbitt in "The Case of the Angry Dead Man." In 1966 he played murderer Harry Lannon in "The Case of the Unwelcome Well." In 1964 he played Ed Pierce in "The Case of the Ruinous Road".

In 1969 he lent his vocal talents to the Walt Kelly/Chuck Jones animated television special "The Pogo Special Birthday Special." Other voice contributors were June Foray and both Chuck Jones and Walt Kelly themselves.

Between 1974 and 1977, Tremayne appeared on the Saturday morning Shazam! television series based on the DC Comics superhero Captain Marvel. In the role of Mentor, Tremayne served as the literal mentor of the programme's protagonist, young Billy Batson.

In 1987, Tremayne appeared on General Hospital as Edward Quartermaine for six months, the oldest character in that series, as a temporary replacement for David Lewis. He played the deceased Victor Lord for one month on One Life to Live during the 1987 Heaven storyline in which daughter Vicki Lord Buchanan (Erika Slezak) was reunited with most every character that had died on the show after a heart attack left her in purgatory.

Tremayne was elected to the National Radio Hall of Fame in 1995.

In 2003, Tremayne died of heart failure at St. John’s Health Center in Santa Monica, California at the age of 90.