Abstract: In order to realize the resource utilization of waste tobacco stems and improve the application status on waste tobacco stem, using waste tobacco stem as fermentation substrate, and the production of Beauveria bassiana as response value, the fermentation process of Beauveria bassiana was optimized using the response surface methodology (RSM).Through Plackett-Burman (PB) design, the most significant factors affecting the production of Beauveria bassiana using waste tobacco stem as fermentation substrate were identified.Based on the results of the Plackett-Burman (PB) experiment, the steepest ascent experiment was used to approach the optimal region, and the mathematical model of optimization of Box-Behnken Design (BBD) response surface was established to determine the optimal conditions.The results showed that when the incubation time was 9.40 days, concentration of peptone and yeast powder was 1.21% and 1.03% respectively, the prediction spore yield of Beauveria bassiana was 1.17×10¹⁰/g, the actual spore yield was 1.13×10¹⁰/g, and the fitting degree was 96.58%.Therefore, the optimal fermentation conditions from RSM are reasonable, and have practical application value in microbial prevention and control and the comprehensive utilization of waste tobacco stems.