In this work, a three-level Box–Behnken factorial design was employed combining with response surface methodology (RSM) to optimize the medium composition for the production of the mycelial biomass and exo-polymer in submerged cultures by Grifola frondosa GF9801. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of mycelial biomass and exo-polymer. The model estimated that, a maximal yield of mycelial biomass (17.61g/l) could be obtained when the concentrations of glucose, KH2PO4, peptone were set at 45.2g/l, 2.97g/l, 6.58g/l, respectively; while a maximal exo-polymer yield (1.326g/l) could be achieved when setting concentrations of glucose, KH2PO4, peptone at 58.6g/l, 4.06g/l and 3.79g/l, respectively. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yields of mycelial biomass and exo-polymer. Maximum mycelial biomass yield of 22.50g/l was achieved in a 15-l fermenter using the optimized medium.