Abstract: In order to explore the utilization of waste tobacco stems, tobacco stems were studied as a fermentation substrate. The effects of particle size and moisture content of tobacco stem on spore yield of Beauveria bassiana were studied via a solid-state fermentation process, and Plackett-Burman (PB) design was used to analyze the related factors influencing the production of Beauveria bassiana, and to optimize the fermentation conditions. The results showed that the particle size of the starting stems was suitable as the main fermentation substrate, the growth and spore germination of Beauveria bassiana were the best when the mass ratio of the stem to water was 1:3.5. Among the 9 selected factors, incubation time, peptone and yeast powder dosage were the key factors affecting the spore yield of Beauveria bassiana. Under PB design conditions, the maximum spore yield of Beauveria bassiana reached 8.59×10⁹/g. This study demonstrated the feasibility of using waste tobacco stem as a fermentation feedstock, and provided a new comprehensive way to utilizing waste tobacco stems.