Abstract: In order to effectively degrade the protein in tobacco leaves and improve the quality of tobacco leaves, bacterial strains capable of degrading tobacco protein were isolated and screened from the surface and inside of tobacco leaves and the rhizosphere soil of tobacco plants, and sprayed on the surface of tobacco leaves before curing. The changes in physiological and biochemical characteristics, appearance quality and sensory quality of tobacco leaves before and after spraying were compared, and the changes in microbial community and gene expression of tobacco leaves before and after spraying were analyzed by amplicon sequencing technology and transcriptomics technology. The results showed that a strain of Bacillus tequilensis YP5 was isolated and screened, and its initial protease activity was 71.05 U/mL. The protein content of tobacco leaves treated with this strain was 2.87 mg/g, which was 36.92% lower than that of the control group (4.55 mg/g). During curing, the activities of peroxidase (POD), superoxide dismutase (SOD) and amylase increased, and those of polyphenol oxidase (PPO) and nitrate reductase (NR) decreased. After curing, the chemical composition in tobacco leaves was coordinated, the appearance quality was better, the proportion of middle tobacco leaves increased, and the overall quality of tobacco leaves was significantly improved. Compared with the control group, the bacterial abundance in the tobacco leaves of the strain YP5 treatment group was significantly increased and the fungal abundance was significantly decreased. Transcriptome analysis showed that the strain YP5 treatment mainly affected the metabolic pathways of phenylpropanoid biosynthesis, oxidative phosphorylation and photosynthesis in tobacco leaves.