Fluorescence emission difference microscopy is a super-resolution imaging technique with strong universality of fluorescent dyes and low phototoxicity. However, due to the limitation of its principle, traditional fluorescence emission difference microscopy has a high system complexity, low stability and limited imaging speed. In order to improve these defects, we design and build a set of multi-color virtual fluorescence difference microscopy system, and it’s imaging method and parameter are analyzed. On the basis of the existing principle of multi-color virtual fluorescence emission difference microscopy, the influence of the signal-to-noise ratio and background is further considered, and a virtual fluorescence emission difference microscopy imaging model that can be verified experimentally is established. The experiments show that the system and method have the characteristics of simple structure, strong background denoising ability, strong universality of fluorescent dyes, and low phototoxicity. Its imaging resolution is 1.9 times higher than that of confocal system, and its imaging speed is doubled compared to the traditional fluorescence emission difference microscopy system. It has obtained good imaging results at three wavelengths, and has been experimentally verified in biological cell imaging.