Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.