Abstract: Condition parameters for anti-microbial photodynamic technology (APDT) of methylene blue (MB) against Enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated. The influence of concentration of MB, illumination time and incubation time on colony-forming unit (CFU) were compared by single factor experiment. The mathematical model describing the relationship between the CFU of O157 and each factor was developed in order to optimize the condition parameters by Box-Behnken center combination experimental design. The optimum values of the above conditions were obtained as:illumination for 27. 66min and incubation for 19. 00min under the presence of 24. 08mg/L MB. Under such conditions, the population of O157 was down to 1. 03logCFU, close to the theoretical prediction value of 1. 00logCFU.