Abstract: In order to improve the smoking quality of the discarded tobacco leaves and enhance the usability of tobacco leaves, three enzyme systems and strains (protein polypeptide enzyme system, lignin enzyme system, and flavor-enhancing strain) were selected for a compound biological fermentation preparation. Taking the degree of quality improvement as the evaluation index, the single-factor method and uniform design method were used to optimize the biological fermentation process of tobacco leaves. The results showed that: 1) The optimum fermentation process conditions were determined as follows: the mass ratio of fermentation preparation to tobacco was 4%, the moisture content of the raw material was 22%, the fermentation temperature was 37 ℃, and the fermentation time was 42 h. 2) Among the factors of fermentation process, the improvement degree of fermentation temperature on quality is greater than that of material moisture and fermentation time. 3) Under the optimal fermentation conditions, the quality of tobacco leaves was improved by 1.04, the impurity and irritation were reduced, and the sensory quality was significantly improved. After fermentation, the contents of protein, starch, pectin and lignin of tobacco leaves decreased by 9.17%, 15.59%, 16.67% and 7.84%, respectively, and the volatile aldehydes, ketones and alcohols increased by 23.70%, 15.33% and 41.03%, respectively, compared with the untreated samples. The chemical composition of tobacco leaves is more coordinated, and the usability of tobacco leaves is improved after treated with the optimal fermentation process.