FM4-64, a kind of membrane-selective fluorescent dye, was utilized to stain themembrane structure of Botrytis cinema and Phytophthora capsici. The effect of FM4-64 on hypha was observed under confocal microscope. The results indicated that the plasma membrane, speta and vesicles of the hyphae could be stained with FM4-64 at the concentrations of 1 to 20 μmol/L. The optimal dye concentration was 6 μmol/L for the both pathogens. At the same concentration of FM4-64, the staining of organelles was in a time-dependent manner and the different organelles were stained in diferrent times. The staining of plasma membrane and vesicles should be completed in 5 minutes, as other organelles can be stained after 10 minutes.