Abstract: The efficacy of anti-microbial photodynamic technology (APDT) against Listeria monocytogenes (LM) biofilm (BF) by methylene blue (MB) under photocatalysis-xenon lamp light (power density 200mW/cm2) was visualized using confocal laser scanning microscope (CLSM) and investigated by counting colony-forming units (CFU) . Obtained results indicated that the efficacy of MB-APDT to LM BF was clearly visual through CLSM based on SYTO9/PI staining. In addition, standard plate counts of cells recovered from biofilms were used to quantify the effects of MB-photoactivated. The inactivation rate of cultured 8h LM BF was over 99.7% by 0.1μg/mL MB and 20min of irradiation, exposure to 50μg/mL MB resulted in almost complete killing. When the concentration of MB was 1μg/mL at the illumination for 5min, cultured 8h BF produced 99.6% inactivation while cultured 36h BF was only 78.7%. The ability of inactivate biofilm bacteria was further enhanced at the illumination for 30min, 99.96% inactivation rate of cultured 8h BF and 99.94% of cultured 36h BF, respectively. The efficacy of MB-APDT to LM BF mainly depends on the concentration of MB and illumination time, and LM BF were effectively inactivated.