Donders' interest in ophthalmology began in 1847 with a study of muscae volitantes, the problem of specks seen floating before the eye. This study resulted in his formulation of what is now known as Donders' law: the rotation of the eye around the line of sight is involuntary.

As professor of physiology at the University of Utrecht (1852–89), Donders did research that immediately improved diagnosis, operative treatment, and use of eyeglasses to correct impairments of vision. He found (1858) that hypermetropia (farsightedness (hyperopia)) is caused by a shortening of the eyeball, so that light rays refracted by the lens of the eye converge behind the retina. He discovered (1862) that the blurred vision of astigmatism is caused by uneven and unusual surfaces of the cornea and lens, which diffuse light rays instead of focusing them. This last finding created the field of scientific clinical refraction.

Donders summarized his studies in On the Anomalies of Accommodation and Refraction (1864), the first authoritative work in the field.