Willy Kühne, the son of a wealthy Hamburg merchant, was educated at the University of Göttingen where he obtained his PhD on induced diabetes in frogs in 1856. He studied further in Jena, Berlin, Paris (under Claude Bernard), and Vienna before joining Rudolf Virchow's Berlin institute in 1861. Kühne later held chairs of physiology, first at Amsterdam from 1868 and from 1871 until his retirement in 1899 at Heidelberg.

Kühne worked with Russell Chittenden on problems of digestion, and he isolated trypsin from pancreatic juice. In 1859, working with the sartorius muscle, he demonstrated that nerve fibers can conduct impulses both ways, and also showed that chemical and electrical stimuli can be used to excite muscle fibers directly.

He also, in the late 1870s, coined the term rhodopsin for the substance, also known as visual purple, first discovered in the retinal rods by Franz Boll in 1876. It was soon realized that the pigment was bleached out of the retina by light and resynthesized in the dark. Kühne realized that this could be used to photograph the eye, to take what he termed an ‘optogram’ by the process of ‘optography’. To achieve this he placed a rabbit facing a barred window after having its head covered with cloth to allow the rhodopsin to accumulate. After three minutes it was decapitated and the retina removed and fixed in alum, clearly revealing a picture of a barred window.

Later investigations of rhodopsin by such scholars as George Wald revealed much about the mechanism of vision.