The fluorescence emission intensity is vital to scientific observation using fluorescence microscopy. Three important factors influencing the intensity of fluorescence emission were theoretical analyzed, including the absorption ability of excitation photons, fluorescence quantum yield, and fluorescence saturation & fluorescence quenching. The authors pointed out that fluorescence molecules with large optical absorption cross section and high quantum yield can effectively guarantee the fluorescence emission intensity, and one also can avoid unnecessary fluorescence saturation if excitation intensity was determined in a reasonable range. Furthermore, fluorescence quenching experiments were studied in ultra-high vacuum (UHV) and atmospheric environment, respectively. We found that fluorescence quenching in UHV was imperceptible, while the fluorescence intensity in the atmosphere decreased exponentially.