In order to achieve multitarget DNA test in microfluidic chip at th e same time,this paper develops a multicolor fluorescence detection system for microfluidic chip based on a uniform illumination structure of light pipe homogenizer.According to the characteristi cs of the polymerase chain reaction (PCR) chamber in microfluidic chip,an orthog onal type fluorescent detection system is designed using super bright white LED modules,light pipe homogenizer,dichroic mirror and CCD.The f luorescent signals of all PCR reaction chambers in microfluidic chip can be collected at the same time,and also a variety of fluorescent markers can be detected simultaneously through the transformation of the optical filter. Fluorescein sodium solution was carried out to test the uniformity of excitation light,the fluorescence image uniformity is 93.99%,and it can meet the design requirements.To evalu ate the performance of the developed fluorescence detection system,we successfully performed PCR analysis of pUC-18.The experimental results show that the DNA concentration change of PCR reaction cha mber is consistent with that of fluorescent signal,and the limit of detection of pUC-18is 0.05pg/uL.The melting curves indicate that there are no primer dimers and specificity is good.The amplification efficiency is 97.28% and can achieve commercial-level.