Two-photon excited fluorescence based on confocal laser scanning system was used to study the cellular localization of 5-ALA induced PpⅨ in DHL cells. Fluorescence probes of rhodamine123, DioC6(3) and LysoTracker Green were used to indicate the cellular localization of 5-ALA induced PpⅨ with a double-labeling method, and real-time image processing was carried out to detect the image edges, extract and segment the information of fluorescence images. By using the simulation of Matlab software, we calculated the correlation coefficient between fluorescence probe image and 5-ALA induced PpⅨ image after a 3-hour incubation time, and the results show r=0.564 for mitochondria, r=0.465 for endoplasmic reticulum and r=0.366 for lysosome. It can be seen that 5-ALA induced PpⅨ mainly distributes in mitochondira, but also in endoplasmic reticulum and lysosome, at least in the lysosome. Two-photon excited fluorescence can be a useful method for quantitative or qualitative cellular localization analysis of 5-ALA induced PpⅨ.