Abstract: In order to clarify the biochemical functions of tobacco endophytic bacteria and explore their application in tobacco leaf fermentation, a strain NS42 with high-efficiency in degrading proteins, cellulose, starch, lignin, and pectin was isolated from fresh tobacco plants. Based on morphological characteristics and phylogenetic tree analysis, NS42 was preliminarily identified as Bacillus velezensis. Following fermentation with NS42, the chemical composition of tobacco leaves was modulated, leading to an enhancement in the aroma quality of flue-cured tobacco leaves. Notably, both the aroma intensity and sweetness of flue-cured tobacco leaves increased, while irritation and offensive odors were diminished, resulting in a more pleasant aftertaste. The reducing sugar content increased by 18.51%. The contents of starch, cellulose, lignin, protein, and pectin in tobacco leaves decreased by 15.61%, 9.67%, 8.49%, 12.70, and 16.47%, respectively. The content of aroma compounds including 5-methylfurfural, β-dihydrodamascenone, megastigmatrienone, farnesylacetone, β-ionone, solanone, phenylethyl alcohol, benzaldehyde, and neophytadiene were significantly elevated. The whole genome of NS42 comprises 6885670540 base pairs encoding 3919 genes, among which 7 genes were related to protein degradation, 6 to cellulose degradation, 6 to starch degradation, 9 to pectin degradation, and 4 to lignin degradation. This strain can utilize various polysaccharides and proteins as carbon and nitrogen sources. In this study, an endophytic Bacillus strain with robust degrading enzyme activity was isolated and characterized, demonstrating a significant impact on the chemical aroma components and sensory quality of tobacco leaves, thereby showcasing substantial potential for enhancing tobacco leaf quality.