Perkins was born in New York City, the son of Janet Esseltyn Rane and stage and film actor James Ripley Osgood Perkins. He attended The Brooks School, Buckingham Browne & Nichols, Columbia University and Rollins College, having moved to Boston after his father's death in 1942.

Perkins' first movie was The Actress (1953); he received an Academy Award nomination for his role in his second film, Friendly Persuasion (1956). After other acclaimed performances both in film and on Broadway, he starred as Norman Bates in the 1960 film Psycho. The role affected the rest of his career.

Following the success of Psycho, Perkins had a successful career in Europe. He created a portrayal of Joseph K. in Orson Welles' The Trial (1962), a cinematic adaptation of the novel by Franz Kafka. Upon returning to America, he took the role of a disturbed young murderer in Pretty Poison (1968). He starred in the sequels and prequel to Psycho, including Psycho II, Psycho III (which he directed) and Psycho IV: The Beginning. He also played the chaplain in Catch-22 (1970). Despite these successes, most of his later work was made-for-TV movies. Perkins also co-wrote, with composer/lyricist Stephen Sondheim, the screenplay for the 1973 film The Last of Sheila, for which they received a 1974 Edgar Award from the Mystery Writers of America for Best Motion Picture Screenplay.

Among his Broadway credits are the Frank Loesser musical Greenwillow (1960) and Bernard Slade's 1979 play Romantic Comedy opposite Mia Farrow. Perkins's life was meticulously documented in the 1996 biography Anthony Perkins: Split Image written by Charles Winecoff.

Perkins was bisexual, having had affairs with a number of men, including 1950s and 1960s film star Tab Hunter, dancer Rudolf Nureyev, composer/lyricist Stephen Sondheim and dancer-choreographer Grover Dale, with whom Perkins had a six-year relationship prior to his marriage to Berry Berenson. He claimed to have been exclusively gay until his late 30s, when he met actress Victoria Principal.

Perkins died in 1992, at age 60 from complications from AIDS. One day before the ninth anniversary of his death, Perkins' widow, Berenson, died on American Airlines Flight 11, the flight that was hijacked by terrorists and crashed into the North Tower of the World Trade Center during the September 11, 2001 attacks. He had two sons: actor Osgood "Oz" Perkins (b. 1974), and musician, Elvis Perkins (b. 1975).

I have learned more about love, selflessness and human understanding from the people I have met in this great adventure in the world of AIDS than I ever did in the cutthroat, competitive world in which I spent my life.