Tooker was raised in the fairly rural atmosphere of Bellport, New York, on Long Island. At the age of seven he began two years of painting lessons with a family friend and neighbor, Malcolm Fraser. He attended local public schools, but the final two years of his secondary education where taken at Phillips Academy, Andover, Massachusetts, from which he graduated in 1938. Andover, although adhering to a strict and conventional curriculum was unusual in offering art instruction and making studio art facilities available to student; Tooker made the most of the opportunity. The next four years he attended Harvard College, Cambridge, Massachusetts, graduating in 1942. Although he immediately entered the armed forces, he was discharged within a few months because of aggrevation of a long-standing stomach ailment.
In the spring of 1943 he entered the Art Students League, New York, for two years of study under Kenneth Hayes Miller, Reginald Marsh, and Harry Sternberg. However, it was his meeting with Paul Cadmus in 1944, and somewhat later with Jared and Margaret French, that had lasting impact on his art. Cadmus was a strong influence on the formal character of Tooker's work (termed "Magic Realist"), but more importantly it was Cadmus who introduced him to the Renaissance technique of egg tempera on gessoed panel, which he has consistently practiced since 1945.
Tooker began to achieve recognition in 1950, when perhaps his most well-known painting, Subway, was acquired by the Whitney Museum of American Art, New York, and, with Cadmus and French, he was selected for inclusion in Lincoln Kirstein's exhibition, Symbolic Realism. His first solo exhibition took place in New York the next year. Thereafter his work was regularly seen in group shows, as well as in periodic solo exhibitions. A major survey of his work was organized by the Fine Arts Museums of San Francisco in 1975
Tooker taught at the Art Students League from 1965 to 1968. He was elected to the American Academy and Institute of Arts and Letters in 1983. Since 1960 he has made his home in Hartland, Vermont, although he maintains a studio on Long Island, and frequently spends time in Spain. Tooker is represented in New York by the Marisa del Re Gallery.
