In order to explore the influence of environmental lighting on visual fatigue during safety sign identification, 12 mixed lighting environments were designed with illuminance and color temperature as environmental variables. Eye movement data were tested under different conditions by eye-tracking technology and a two-factor ANOVA was conducted. Combined with the test results of reaction speed and comfort, the changes of visual fatigue under different lighting conditions were analyzed. The experimental results show that the change of illumination has a significant effect on visual fatigue, subjects are more prone to visual fatigue in low illumination environment; The change of color temperature has little effect on visual fatigue, but in different mixed lighting environments, the change of color temperature will affect the visual comfort of the subjects, and the fatigue state will change accordingly. The suitable color temperature range for the lighting conditions in the working environment is 2 100 ~ 3 500 K, and the illuminance range is 550 ~900 lx. It is concluded that companies should pay attention to improving the lighting conditions during production, avoid reducing the recognition efficiency of safety signs by workers due to visual fatigue, and ensure the safety and health of workers.