Abstract: Alternaria longipes is the causal agent of tobacco brown spot, a world-wild tobacco disease. Based on Czapek medium supplemented with CaCO3 and apple cutin, the liquid fermentation conditions were optimized in order to acquire higher cutinase activity. Optimum culture time, temperature and pH were 8 d, 25℃ and 5.5, respectively; and optimum carbon and nitrogen sources were dextrose and maltose instead of NaNO3. According to this fermentation and orthogonal test, the optimum composition of culture medium included dextrose 3％, casein 1％, Tween-80 0.3%. A 11.0-fold higher cutinase activity was obtained compared to the basal medium and the enzyme activity was enhanced from 15.8 U/mL to 175.2 U/mL.