Abstract The non-uniformity of image directly affects the application of EMCCD in various disciplines. The proposed method can significantly improve the uniformity of EMCCD output image. The correction algorithm of "reverse split and forward recovery" is determined through analyzing the imaging model of EMCCD, and the comprehensive non-uniformity correction function model is established. The 8-tap EMCCD chip CCD220 of British e2v company is used for experimental verification. The results show that after the comprehensive correction the consistencies of the light response characteristic curve and the multiplication gain curve of each channel in EMCCD are obviously improved, and also the photo response non-uniformity (PRNU) of the output image is substantially reduced from 24.5 to 4.1%, which prove the effectiveness of the proposed method.