Abstract: Clostridium botulinum and its spores were the primary sterilization objects in vacuum packaging, frozen foods and canned foods due to its strong resistance and rapid growth, and become one of the main causes of food poisoning. The effects of the addition concentration of spore germination agents L-alanine and KCl, treatment temperature and time were used to study on influence of the germination conditions of C. botulinum spores by using the single-factor experiments and response surface analysis. To explore the optimal conditions that affect the germination of C. botulinum spores, and then combined with normal pressure 100 ℃, 20 min sterilization, to study the final killing effect of C. botulinum. The results showed that the processing temperature was 75.5 ℃, the processing time for 25.0 minutes, the concentration of L-alanine added was 15.4 mmol/L, KCl concentration was 0.53%, and then germination rate of spore was up to 89.91% ± 1.31%. After two times of sterilization at 100 ℃ for 20 min, the killing rate of C. botulinum could reach 98%. The research results could provide a certain technical guidance for the normal temperature sterilization of canned food in the food industry.