In combination of reference standard sampling and calibration methods, a multi-target area calibration linear correction algorithm was proposed to correct the nonuniformity of fluorescence detection of microfluidic Polymerase Chain Reaction(PCR) system and to improve the performance of the system. The fluorescein sodium solution with similar spectral characteristics to PCR fluorescence marker SYBR Green was used as the test sample, and the emission fluorescence intensities of eleven uniformity sodium fluorescein solutions with different concentrations were measured. The linear relationship between fluorescence intensities and fluorescein sodium concentrations was analyzed and the correction coefficient matrix in each imaging target area on a CCD was calculated by a two-point linear correction algorithm. The results after correction show that the imaging uniformities in three different concentrations of sodium fluorescein solutions are improved respectively from 71.28%, 72.01%, 70.73% to 77.49%, 80.07%, 90.64%. The relative standard deviations of Ct value for the same concentration DNA template are reduced respectively from 4.38%, 1.94%, 3.31% to 2.44%, 0.79%, 1.31%. These results indicate that the proposed nonuniformity correction algorithm significantly improves the accuracy of microfluidic real-time PCR.