A visual comfort evaluation method of video display terminal (VDT) based on pupil diameter was proposed. An eye movement experiment was conducted in three kinds of lighting and five kinds of video display terminal luminance, in which the pupil diameter data of the subjects were collected, and the subjective ratings were also given for visual comfort under each condition. Statistical analysis was used to systematically study the corresponding relationship between pupil diameter change and visual comfort from three aspects, including gender, eyes habit, and whether glasses-worn or not. Experimental results showed that, compared with the pupil diameter under the condition of visual comfort, the relative variation of pupil diameter is from 17.61% to 24.55% when the visual discomfort is caused by the dark light environment, the relative variation of pupil diameter is from -6.12% to -12.45% when the visual discomfort was caused by the bright light environment. But the relative variation of pupil diameter is from -2.14% to 2.14% when the subjects were in a state of visual comfort. This work is of great significance for the establishment of VDT visual health assessment standards and the design of healthy light environment.