Abstract: In the present study, a strain of Grifola frondosa LT was used to evaluate the effects of medium nutrition on the growth of mycelium and production of extracellular polysaccharides. Glucose, peptone, monopotassium phosphate and vitamin B1 were established as the optimal carbon source, nitrogen source, inorganic salt and growth factor via single factor test. The concentration of medium nutrition was further optimized through multi-factor orthogonal test, and the optimum fermentation condition for the growth of mycelium were 10% of bean sprouts 200 mL, glucose 20 g/L, peptone 5 g/L monopotassium phosphate 5 g/L and vitamin B1 30 mg/L, and the optimum fermentation condition for production of extracellular polysaccharides were 10% of bean sprouts 200 mL, glucose 20 g/L, peptone 3 g/L monopotassium phosphate 4 g/L and vitamin B1 30 mg/L. This research was of certain values to further fermentation researches and applications of Grifola frondosa mycelium and polysaccharides.