T24 cells are incubated with a novel photosensitizer chlorophyllin f, and then irradiated by 650 nm laser light. MTT assay is used to detect the growth inhibition rate of T24 cells. The apoptosis of T24 cells is observed by flow cytometry. The Mito Tracker Green probe is used to label mitochondrion, and the confocal laser scanning microscopy excited by 488 nm and 405 nm dual channels is applied to reveal intracellular localization. With 4 J/cm2 of optical energy density, the growth inhibition rates of 2.5, 5 and 10 μg/mL chlorophyllin f are 17.68%, 49.35% and 84.42%, while with 1 J/cm2 optical energy density, the growth inhibition rates are 4.34%, 37.42% and 78.38% respectively. The apoptosis rate of treatment group is (45.23±1.2)%, significantly higher than control groups. The confocal laser scanning microscopy reveals that, the red fluorescence emitted by chlorophyllin f and green fluorescence emitted by Mito Tracker Green probe distributes almost the same. Therefore, the photodynamic effect of chlorophyllin f on T24 cells is significant. Chlorophyllin f mainly locates and functions at mitochondria, and induces apoptosis, which may be one of the mechanisms against T24 cells.