[Objective] To optimize the fermentation medium of an antitumor endophytic fungus isolated from Ginkgo biloba. [Methods] In this study, the dry weight of mycelia, weight and antitumor activity of crude extract were used as standards to screen out the best carbon source and nitrogen source through single factor experiment. Then the culture medium recipe was optimized by the orthogonal test in five factors and four levels. [Results] The best culture medium recipe was: glucose 45 g/L, peptone 8 g/L, K2HPO4 0.5 g/L, MgSO4·7H2O 0.2 g/L, KCl 1 g/L, FeSO4 0.01 g/L. After optimization, the dry weight of mycelia, weight and antitumor activity of crude extract were increased by 41.88%, 226.52% and 19.31% respectively. [Conclusion] The production and antitumor activity of crude extract was significantly increased after optimization, which might be beneficial to reduce the fermentation scale and workload in scale up fermentation later.