In order to analyze the visual fatigue degree of the foreground and background color combination of the display of civil aircraft cockpit, the visual afterimage experimental paradigm is used in the present study. Objective eye movement data and subjective reported visual afterimage color were collected. The difference of pupil diameter under different color combinations showed that background color had a significant effect on visual fatigue. In addition, black background color can lead to the lowest degree of visual fatigue, followed by brown background color. Moreover, blue background color was the most likely to cause visual fatigue. The subjective report data showed that the visual afterimage color was related to the foreground and background color combination. In the combination of background color and foreground color, the ratio of visual afterimage color and background color is the highest, followed by the foreground color, and the complementary color of foreground color or background color. Based on the above results, the color design suggestions of civil aircraft cockpit interface are proposed in this work.