Rushton, the son of a London dental surgeon, studied medicine at Cambridge University and University College Hospital, London. He worked in Cambridge from 1931 until 1968, being appointed professor of visual physiology in 1966. In 1968 he moved to the Florida State University, Tallahassee, to serve as research professor of psychobiology until his retirement in 1976.

Rushton studied the theory of nerve excitation until the early 1950s but changed to studying visual pigments following some work with Ragnar Granit in Stockholm. Rushton's novel technique was to shine light into the eye and measure the amount reflected back with a photocell. The effects of rhodopsin, the ‘visual purple’ of Willy Kuhne, could be discounted by working with the fovea, the retinal region of sharpest vision devoid of rhodopsin-containing rods. As the fovea is also deficient in blue cones, Rushton argued that by limiting pigment absorption measurements to this small area the properties of the red and green cones alone should be revealed.

By examining color-blind individuals, Rushton showed that red-blind defectives lack the red-sensitive pigment erythrolabe and that people who cannot distinguish between red and green lack the green-sensitive pigment chlorolabe.