A design method for night lighting system of tomato picking robots based on experiments design was presented. In the design of experiments, an image segmentation evaluation index, which could determine the optimal night lighting system, was proposed. It was calculated as the ratio of F between intraclass and interclass variances of foreground and background of tomato images captured at night. Three factors, including light source type, light source layout and image acquisition distance, were considered. Moreover, orthogonal experiment table L18(6×36) was used to arrange experiments. Testing results showed that, the light source type and light source layout were two significant factors for night lighting system, while the image acquisition distance was not. So, it is focused on the influence of light source type and light source layout in the night lighting system. Meanwhile, the F value of fluorescent lamp was 2.159 which was the highest among those values of six types of light source type, and that of the diagonal layout was 2.234 which was the highest among the values of three types of light source layout. Therefore, in the current trial, the optimal light was fluorescent lamp, and the best light source layout was diagonal layout for the night lighting system of tomato picking robot. F values of tomato images captured at night were compared with image segmentation results using the OTSU algorithm based on normalized R-G color difference. Comparative results showed that the image segmentation results were better for images with higher F. The image segmentation evaluation index based on F and the design method for night lighting system are effective.