Johnson joined the Army Air Corps in World War II and served as a gunner on bombers. He had a very distinguished record and was highly decorated for his service.

He flew 44 combat missions as a crewman in B-24 Liberator bombers, and won seven decorations, including a Purple Heart for injuries sustained when his plane was shot down over the Philippines. When the war ended, he joined the Army Reserves, and used the GI Bill to fund his acting studies.

He became a close friend of Audie Murphy and later appeared with him in one of his films. Johnson's Hollywood career began in 1952, with the college fraternity hazing exposé For Men Only. His early roles were primarily in westerns and science fiction such as It Came from Outer Space (1953), This Island Earth (1955), Attack of the Crab Monsters (1956), and The Space Children (1958). He also appeared in a Ma and Pa Kettle vehicle, Ma and Pa Kettle at Waikiki (1955), and attempted to prevent the assassination of Abraham Lincoln in the Twilight Zone episode "Back There". He also appeared as a Professor {!} in Twilight Zone episode "Execution". {Both episodes featured Time Travel}.

He is best known to the public as The Professor, the erudite polymath who can build all sorts of inventions out of the primitive materials on the island, but can't find a way for the castaways to get rescued. Gilligan's Island was in first-run from 1964 to 1967, but has been shown in reruns continuously since that time. Johnson was typecast in this sort of role, but he has willingly participated in all reunion movies, cartoons, and other events related to the program. When recognized as "The Professor" in public, he is gracious and friendly, and doesn't object to being addressed as "Professor".

Johnson was asked to take off his shirt when auditioning for the "Gilligan's Island" role, he refused but still got the job.

Before accepting the role of "Roy Hinkley", he made "Gilligan's Island" producer Sherwood Schwartz promise him that when he made scientific statements that they would be accurate.

After Gilligan's Island he appeared in several other movies and television shows, perhaps most notably the miniseries Vanished, based on a novel by Fletcher Knebel (1971), uncredited in the Robert Redford spy thriller, Three Days of the Condor (1975), and on the NBC soap opera Santa Barbara.

An episode of the show Newhart featured the Beavers (the men's organization) watching a Gilligan's Island marathon on TV. When they are suddenly evicted from the room, one of the Beavers protests, "I want to see how it ends!" He is assured that they (the Castaways) don't get off the island. Russell Johnson portrayed the protesting lodge member.

His son David ran the AIDS program for Los Angeles, California until his death of AIDS in 1994. Johnson has been a full-time volunteer for AIDS research fundraising since his son was diagnosed and in the years since his death.

Johnson has written his memoirs of the show, a book titled Here on Gilligan's Isle.

He currently lives in Bainbridge Island, Washington.