To sdudy the non-visual effect to human based on the analysis of pupil contraction under low illumination LED light, ten subjects were selected to conduct pupillary light reflex test under different conditions of six kinds LED light with different colors, five kinds of low illumination. All these tests were recorded by infrared video recorder and the pupil size was calculated by the program based on OpenCV. The result shows that steady pupil contraction rate and recovery pupil contraction rate will be larger as the migration from long wave to short wave light source under the same illumination. And the non-visual effect of blue light source is the most significant. Also, steady pupil contraction rate and recovery pupil contraction rate will be larger as the illumination increased under the same light source. And there is a linear relationship between these two pupil contraction rates under the illumination of 10-70 lx with the correlation coefficient of more than 0.95. Two pupil contraction rates have a linear relationship between circadian factor, which shows that the pupil contraction rate could well represent the degree of non-visual biological effect.