[Objective] We optimized degradation conditions for diethylstilbestrol (DES) by Aspergillus oryzae M-4. [Methods] Medium components and culture conditions were screened by using Plackett-Burman method. The degradation conditions were then optimized through Box-Bohnken design. [Results] The optimal medium was composed of 1.3% peptone, 0.045% CaCl2, 0.5% glucose, 0.15% K2HPO4, 0.05% KH2PO4, 0.05% NaCl, 0.2% Tween 80 and 44 mg/L diethylstilbestrol. The optimal culture conditions were as follows: initial pH 7.5, inoculum age 72 h, rotation rate 140 r/min, culture temperature 28 °C, culture time 72 h. [Conclusion] Under the optimal conditions, the degradation rate of diethylstilbestrol was 83.89%, 1.38-fold over the value before optimization (60.98%), showing a significant difference (p<0.01).