Karloff was born William Henry Pratt at 36 Forest Hill Road, East Dulwich, London, England where his blue plaque can be seen. He was brought up in Enfield. His paternal grandmother was Eliza Julia (Edwards) Pratt, a sister of Anna Leonowens, whose tales about life in the royal court of Siam (now Thailand) were the basis of the musical The King and I. Her maternal grandmother was of East Indian origin from Calcutta in Bengal.

Once Karloff arrived in Hollywood, California, he made dozens of silent films, but work was sporadic, and he often had to take up manual labour, such as digging ditches and driving a cement truck, to pay the bills. His role as the Monster in Frankenstein (1931) made him a star. A year later, he played another iconic character, Imhotep, in The Mummy.

The five foot eleven, brown-eyed Karloff played a wide variety of roles in other genres besides horror. He was memorably gunned down in a bowling alley in the 1932 film Scarface. He played a religious WWI soldier in the 1934 John Ford epic The Lost Patrol.

Karloff gave a string of lauded performances in 1930s Universal horror movies, including several with his main rival as heir to the horror throne of Lon Chaney, Sr., Bela Lugosi, whose rejection of Karloff's role in Frankenstein made Karloff's subsequent career possible. Karloff played Frankenstein's monster three times; the other films being Bride of Frankenstein (1935) and Son of Frankenstein (1939), which also featured Lugosi as the demented Ygor.

Karloff would revisit the Frankenstein mythos in film several times after leaving the film role of the creature. The first would be as the villainous Dr. Niemann in House of Frankenstein (1944) where Karloff would be famously contrasted against the then more popularized Glenn Strange who became the standardized interpretation of the Monster during the 1940s, 1950s and 1960s.

Karloff returned to the role of the "mad scientist" of Frankenstein mode in 1958's Frankenstein 1970 as Baron Victor Von Frankenstein II, the grandson of the original inventor. The final twist reveals the crippled Baron has given his own face (i.e., "Karloff's") to the Monster. The actor appeared at a celebrity baseball game as the Monster in 1940, hitting a gag home run and making catcher Buster Keaton fall into an acrobatic dead faint as the Monster stomped into home plate.

For a fantasy sequence in The Secret Life of Walter Mitty, director Norman Z. McLeod filmed a sequence with Karloff in the Monster make-up, but it was deleted. The final time Karloff donned the headpiece and neck bolts was 1962, for a Halloween episode of the TV series Route 66, but he was playing "Boris Karloff" who, within the story, was playing "the Monster."

While the long, creative partnership of Boris Karloff and Bela Lugosi never led to a close mutual friendship, it produced some of each actor's most revered and enduring productions, beginning with The Black Cat. Follow-ups included Gift of Gab (1934; not horror, but a whimsical comedy featuring cameos from contract stars), The Raven (1935), The Invisible Ray (1936), Black Friday (1940), You'll Find Out (also 1940), and The Body Snatcher (1945), which many believe contains Karloff's greatest performance. During this period he also starred with Basil Rathbone in Tower of London (1939).

In contrast to the characters he played on screen, Karloff was known in real life as a very kind gentleman who gave generously, especially to children's charities. Karloff was also a charter member of the Screen Actors Guild, and was especially outspoken regarding working conditions on sets (some extremely hazardous) that actors were expected to deal with in the mid-1930s. He married six times.

An enthusiastic performer, he was able to return to the Broadway stage in the original production of Arsenic and Old Lace in 1941, in which he played a homicidal character enraged to be frequently mistaken for Karloff. Although Frank Capra cast Raymond Massey in the 1944 film, (which was shot in 1941, while Karloff was still appearing in the role on Broadway) Karloff reprised the role on television with Tony Randall and Tom Bosley in a 1962 production on the Hallmark Hall of Fame.

Somewhat less successful was his work in the J. B. Priestley play The Linden Tree. He also appeared as Captain Hook in the play Peter Pan with Jean Arthur, and in the process revealed a surprisingly good singing voice[citation needed]. He was nominated for a Tony Award for his work opposite Julie Harris in The Lark, by the French playwright Jean Anouilh about Joan of Arc, which was also reprised on Hallmark Hall of Fame.

In later years, Karloff hosted and acted in a number of television series, most notably Thriller, Out of This World and The Veil, the latter of which was never broadcast and only came to light in the 1990s. In the 1960s, Karloff appeared in several films for American International Pictures, including Comedy of Terrors, The Raven and The Terror, the latter two directed by Roger Corman, and appeared as the very brave "retired horror film actor" Byron Orlok (a lightly-disguised version of himself) in Peter Bogdanovich's critically acclaimed 1968 film Targets, which was one of Karloff's final film appearances.

During the 1950s Karloff appeared on British TV in the series "Colonel March of Scotland Yard", in which he solved apparently impossible crimes.

On The Red Skelton Show, Karloff guest starred along with horror actor Vincent Price in a parody of Frankenstein with Red Skelton as the monster "Klem Kadiddle Monster". Karloff also appeared with Robert Vaughn and Stefanie Powers in the spy series The Girl from U.N.C.L.E. in the episode, entitled, "The Mother Muffin Affair" in which Karloff performed in drag.

In the mid-1960s, Karloff gained a late-career surge of American popularity when he narrated the made-for-television animated film of Dr. Seuss' How the Grinch Stole Christmas and provided "the sounds of the Grinch". (The song "You're a Mean One, Mr. Grinch" was sung not by Karloff, but by American voice actor Thurl Ravenscroft, the voice of Tony the Tiger.) Karloff later won a Grammy award in the spoken word category after the story was released as a record.