Abstract: To investigate the effects of different enzyme preparations on the aroma components of extracts from waste tobacco dust, waste tobacco dust was used as the raw material and treated with protease, cellulase, pectinase, ligninase, and amylase. The resulting extracts were systematically analyzed by combining gas chromatography-mass spectrometry (GC-MS), odor activity value (OAV), orthogonal partial least squares-discriminant analysis (OPLS-DA), and sensory evaluation. The results indicated that all enzymatic treatments increased the total aroma component content in the extracts by 25% to 73% compared with the control. Among them, the protease and cellulase treatments exhibited better performance, with ketone content increasing by 88.87% and 85.38%, respectively. Based on OAV and OPLS-DA analyses, key differential aroma compounds, including megastigmatrienone, damascone, solanone, trans-β-ionone, and β-dihydrodamascone, were identified. The sensory evaluation indicated that enzyme-treated extracts contributed to enhancing tobacco, floral, sweet, and roasted notes, improving aroma harmony, and increasing the smoothness and delicacy of smoke, with the protease-treated group showing the best overall performance. Overall, enzyme-assisted treatment improved the aroma composition of extracts from waste tobacco dust and their sensory performance in cigarette flavoring, with the protease-treated sample showing potential for further development and application as a tobacco-derived flavoring.