To improve the liquid fermentation biomass and obtain regular ages and stable characteristics of tobacco stalks lignin degrading strain Trametes hirsuta S13, the fermentation medium and cultivation conditions were optimized for T. hirsuta S13 in flasks used orthogonal and single factor analysis test. The optimum fermentation medium of T. hirsuta S13 contains sucrose 20 g/L, corn flour 20 g/L, soybean meal 60 g/L, MgSO₄0.5 g/L, K₂HPO₄ 1 g/L,KCl 0.5 g/L. The optimal cultivation conditions was as following: temperature 30℃, initial pH 5.0, inoculum amount3 fungus plugs, 180 r/min, broth content 80 mL/500 mL flask. Under the optimal fermentation medium and conditions, the biomass of T. hirsuta S13 was 49.83%（V/V）. The liquid fermentation technology can provides experimental basis for industrial application.