The non-uniform illumination of high-throughput microfluidic qPCR is significant for improving the precision of DNA quantitative results. According to the theoretical principle of qPCR, theoretical analysis was presented. The analysis focused on the deviation of cycle threshold (Ct) resulting from non-uniform illumination. On the basis, the deviation formula of Ct caused by non-uniform illumination was proposed. Moreover, the requirement of excitation light was determined, and the relative standard deviation of excitation should be less than 11.56%. Then, according to the characteristics of microfluidic chip and the requirement of the excitation light uniformity, an illumination system based on light pipe was designed. The relative standard deviations of irradiance, obtained from simulation and experiment, were respectively 3.10% and 6.01%, both less than 11.56%. And the uniform illumination system is able to meet the requirement of high-throughput microfluidic qPCR.