Dark field illumination in high throughput colony selection device is reported in this paper. It improves the contrast of image in the premise of uniform illumination. Three important factors including angular distribution of light radiation, number of LED circle array and distance between the target and the LED circle source were analyzed, which affect illumination uniformity of dark field illumination. The simulation result shows that the illumination uniformity is 93.16% when the angular distribution of light radiation is 75°, the number of LED circle array is 3, and the distance between the target and the LED circle source is 61mm. In addition, Experimental system was set up in order to test designed dark field illumination, which matches with the project requirement and simulated results very well.