Because of the increase in green lighting in recent years, scholars have been trying to find more comfortable lighting methods in various fields to meet people’s lighting needs. In previous studies, we found that most museum lighting was conducted in the form of subjective questionnaires, but in this study, we tried to introduce a new way to explore the impact of the lighting environment on comfort, namely eye tracking technology. This paper aims to explore the influences when viewing paintings in cold, warm, and middle tones under illumination of 50 lx, 150 lx, and 300 lx, respectively, on the visual comfort of viewers, and the use visual fatigue as the evaluation index to find the most appropriate illumination value for different painting systems in the art museum. By collecting eye movement data under different illuminance and color combination of different paintings and subjective evaluation from the subjects, this paper studies the impact of different illuminances and colors on the subjects’ visual fatigue. By considering the illumination intensity of the light environment and the tone of the painting, it can be found that the warm tone painting was more suitable for 150 lx, the cold tone painting was more suitable for 50 lx, and the middle tone painting was more suitable for 300 lx.