Abstract: To investigate the effects of microbial enzyme preparations on aroma formation, change rules and their influencing mechanisms during tobacco leaf enzyme treatment, an enzyme preparation prepared with Stenotrophomonas maltophilia was sprayed onto the tobacco strips of the threshing and redrying line. The dynamic changes in alkaline protease activity, α-amylase activity and contents of protein, starch and volatile aroma components were monitored during the fermentation from 0 to 6 h, and the timing relationships among these factors was analyzed by cross-correlation function (CCF). The results showed that both activities of alkaline protease and α-amylase reached their peaks after fermentation for 3 h. The contents of protein and starch decreased by 18.91% and 30.42%, respectively, compared to 0 h, and the contents of reducing sugar and total sugar increased by 2.477 and 2.263 percentage points, respectively, providing precursors for aroma generation. The aroma peak lagged approximately 2 h behind the enzyme activity peak and was basically synchronized with the degradation of protein and starch. The formation of most aroma components lagged approximately 2 h behind the increase in contents of reducing sugars and total sugar. The changes in sugar-nicotine ratio, reducing sugar-to-total sugar ratio, reducing sugar and total sugar occurred approximately 2 h earlier, and could serve as positive predictive indexes for floral, woody and roasted nutty scents. Therefore, precise control of exogenous enzyme preparation treatment time significantly enhances the contents of volatile aroma substances, thereby improving tobacco leaf quality.