The influences of luminous environment on visual responses and fatigue sensation in art galleries were examined in this study. Field measurements and survey were performed in three galleries under various illuminance and luminous conditions. Result implies that vertical illuminance on art paintings needed to be somewhere between 200 lx and 300 lx to avoid visual discomfort. The vertical illuminance difference between consecutive paintings should not exceed 100 lx to keep visually comfortable environment. Visual comfort sensations were related to glare, visual stimulus, reflection from paintings, illuminance variation, and the sensation that subjects can clearly see the paintings. The sensation of fatigue was significantly influenced by the changes of luminous element that caused visual stimulus and discomfort. The sensation of eye fatigue among physical fatigue was primarily influenced by the glare, visual stimulus and comfort. The psychological fatigue was also influenced by eye fatigue, visual comfort, brightness and satisfaction with color of light.