Abstract: Factorial design and response surface techniques were used to design and optimize the process to increase the yield of trichodermin produced by the Trichoderma harzianum, an endophytic fungus isolated from Llex cornuta. Initially, Plackett-Burman design was undertaken to evaluate the effects of the six factors and three statistically significant parameters including glucose, fermentation time and inoculum size were selected. In the second phase of the optimization process, a response surface methodology was used to optimize the above critical factors, and to find out the optimal concentration levels and the relationships between these factors. By solving the model, the optimal batch fermentation conditions were determined. Under conditions of glucose, 42.8 g/L; inoculum size, 1.39 ml; fermentation time, 102.88 h, the yield of trichodermin reached 147.44 mg in 1 L fermentation liquor. After five batches cultivation, the predicted values were verified by validation experiments.