Abstract: Bacillus subtilis was as the research object, with the killing log value as a measure indicator.Three inactivation parameters including pressure, temperature and pressure holding time were optimized using response surface methodology (RSM) .Box-Behnken design was applied to evaluate the effects of three indepent variables on the inactivation of Bacillus subtilis spores.The simulated quadratic polynomial regression equation of prediction model was set up.The optimum process parameters for inactivation of Bacillus subtilis were obtained as follows:pressure 30Mpa, temperature 48℃, pressure holding time 90min.Under the condition of killing log value was 5.22, compared to the theoretical value, the relative error of 2.32%.The adequacy of the model equation for predicting the optimum response values was verified effectivelu by the validation data.