The cultivation conditions of Pholiota squarrosa mycelium were optimized in order to enhance product efficiency and shorten cultivation cycle. The one-factor experimental design and Plackett-Burman design showed that the optimum carbon source was maltose, the optimum nitrogen source was yeast, the optimum pH was 5, and that the optimum temperature was 23 ℃. What was more, the main factors influencing mycelium growth were maltose, yeast and K_2HPO_4. The optimum medium for Pholiota squarrosa mycelium growth was identified as 35 g/L maltose + 1.5 g/L yeast +0.5 g/L K_2HPO_4+0.5 g/L KH_2PO_4+0.5 g/L MgSO_4 by the experiments of the steepest ascent and orthogonal designs. On the optimum medium the colony diameter was increased from 6.11 cm to 7.41 cm.