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Liquid fermentation could yield substantial mycelia mass and valuable secondary metabolites in large-scale production within a short, fermented duration. The liquid fermented process of mycelia of Poria cocos was optimized using a combination of single-factor experimentation and response surface methodology (RSM) to obtain more extract of P. cocos. The optimal conditions were determined as follows: The carbon source concentration at 1%, the nitrogen source concentration at 1%, the inoculum volume at 7% and a culture time of 9 d. Under these conditions, the ethyl acetate extract mass of P. cocos mycelia reached 0.0577 ± 0.0041 mg. There were significant interactions between nitrogen source concentration and cultivation time. The predicted values by the mathematical model based on the response surface analysis showed a close agreement with experimental data.