Abstract: To investigate the formation and variation patterns of aroma compounds during enzymatic treatment of tobacco leaves and the underlying mechanisms, enzyme preparations derived from Stenotrophomonas maltophilia were sprayed onto tobacco strips on the threshing and redrying line. The dynamic changes in alkaline protease activity, α-amylase activity, protein, starch, and volatile aroma components were monitored during the 0–6 h enzymatic treatment period, and cross-correlation function (CCF) was employed to analyze their temporal relationships. The results showed that both alkaline protease and α-amylase activities reached their peak values at 3 h of enzymatic treatment. Compared to 0 h, protein and starch contents decreased by 18.91% and 30.42%, respectively, while reducing sugar and total sugar contents increased by 2.84% and 2.26%, respectively, providing precursors for aroma formation. The peak of aroma compounds lagged approximately 2 h behind the peak of enzyme activities, and synchronized basically with the degradation of protein and starch. The formation of most aroma components lagged about 2 h behind the increase in reducing sugar and total sugar contents. The sugar-nicotine ratio, reducing sugar-to-total sugar ratio, reducing sugar, as well as total sugar, with a lag of approximately 2 h, could serve as positive predictive indicators for floral, woody, and roasted-nutty aromas. Therefore, precise control of the treatment duration with exogenous enzyme preparations can significantly enhance the content of volatile aroma substances and improve tobacco leaf quality.