Abstract: Response surface methodology was adopted to optimize pulsed light parameters for lethal effect of bacillus subtilis.On the basis of single factor experiments, through the response surface experiment design, pulse voltage, pulse frequency, irradiation distance were selected as the response factor, and the fatality rate was investigated.The results showed that the lethal effect of pulse light on bacillus subtilis was extremely significant ( p < 0.01) , according to the optimal conditions and combined with the actual operation, the optimized parameters were pulse voltage of 2450 V, pulse number of 65, distance of 5 cm, the fatality rate can reach 99.95%, and the order of the influencing factors was A ( pulse voltage) > C ( distance) > B ( pulse number) , and that can be the basis of bacillus subtilis existed in the preservation and the processing of food is inactivated in the future.