Abstract: In this study, the effect of different concentrations of inorganic salts and vitamins on the mycelial growth rate of Phlebopus portentosus wild strain PH-21 was investigated, and then the growth medium was optimized using Box Behnken response surface design, and the activity of bioactive metabolites from mycelial cultures of the strain on different media was evaluated. The results showed that the predicted optimal addition of FeSO₄, CaSO₄ and MnSO₄ were 1.28 mg/mL, 1.10 mg/mL and 0.81 mg/mL respectively, and optimum concentration of vitamins were 128.5 μg/mL of VB₁, 95.1 μg/mL of VB₂ and 94.8 μg/mL of VB₃. The results of validation experiment were consistent with the predicted values of the model. Moreover, the contents of polysaccharides and triterpenoids extracted from mycelia were increased by 61.4% and 53.5% in the optimized medium compared to the control, respectively. Furthermore, DPPH and ABTS free radical scavenging rates increased by 89.2% and 44.6%, respectively. Meanwhile, the content of malondialdehyde（MDA） in cells was significantly reduced, while alleviate the attenuation of superoxide dismutase（SOD）, peroxidase（POD）, and catalase（CAT） activities. Comparing with the PDA medium, adding appropriate concentration of inorganic salts and vitamins to the medium could significantly enhanced mycelium growth and delayed hyphal aging.