Abstract: In order to obtain microbial strains that efficiently degrade tobacco proteins, microbial resources with industrial application value were explored. In this study, we used Bacillus pumilus ZG7 as the starting strain, and the modified error-prone PCR technique was used for the mutagenesis selection of ZG7 strain, firstly, the positive mutant strain was screened by the transparent circle method, then the protease activity of the positive mutant strain was quantitatively determined by the forintol method, characterized the expression levels of different proteases using SDS-PAGE, and then the optimal fermentation conditions of the mutant strain were clarified by the response surface method, and the mutant strain's effect on the degradation of tobacco leaf protein and the sensory quality of the treated leaf was investigated. The degradation effect of the mutant strain on the protein of tobacco leaves (Yunnan Tobacco 87) and the sensory quality effect of the treated tobacco leaves were investigated. The results showed that the high-quality protease-producing mutant strain B. pumilus ZG7-1 had a protease activity of 168.33 U/mL, which was 213.81% higher than that of the starting strain. The mutant strain ZG7-1 significantly enhances the expression of proteases with molecular weights of approximately 28 kDa and 58 kDa, and additionally produces a protease with a molecular weight of approximately 72 kDa. Based on the one-way test, the enzyme production conditions of the mutant strain ZG7-1 were optimized by response surface test, and the optimal protease production conditions of the mutant strain ZG7-1 were determined as follows: sucrose 18.60 g/L, magnesium chloride 3.48 g/L, and inoculum quantity 4.81%, under which the fermentation enzyme activity could reach 491.50±3.06 U/mL, which was 2.92 times higher than that before the optimization. By treating tobacco with bacterial solution of ZG7-1 mutant strain, the protein degradation rate reached 27.05%, and the sensory quality of tobacco was significantly improved. The study showed that the mutant strain ZG7-1 has strong protease production ability and high genetic stability, which can efficiently degrade tobacco protein and improve the quality of tobacco, and has a better application prospect.