Abstract: In order to explore the effect of added enzyme preparation on fermentation of flue-cured tobacco, an experiment on degradation of starch and protein in tobacco leaves was conducted by using various treatments of added enzyme level, time, RH and temperature. The results showed that single application of neutral protease could not achieve favorable degradation rate of protein in flue-cured tobacco, while the best combinations of α-amylase and glucoamylase were found for different stalk position leaves. Under the relatively optimum conditions, three grades tobacco leaves treated by a-amylase, glucoamylase and neutral protease subsequently exhibited degraded starch and protein, significantly increased amino acid, and no changes of reduced sugar, total nitrogen and nicotine.