Antrodia camphorata is a uniquely preserved fungus that is indigenous to and found only in Taiwan. It only grows on a species of rainforest tree that is also native to Taiwan. A. camphorata has been used in traditional Chinese medicine for more than 100 years, and its main effective component are polysaccharides. A culture medium for increased polysaccharides was developed in this study. Soluble starch, wheat bran, and MgSO4 are the key factors of the culture medium based on the results obtained from the Plackett-Burman experimental design. The optimal concentration range of the three factors was examined by the steepest ascent path. Response surface methodology (RSM) was used to optimize the culture medium. A Box-Behnken design was used for the experimental design and analysis of the results to obtain the optimal culture medium. The 3D RS and contour plots derived from the mathematical models were applied to determine the optimal culture medium. The optimum culture medium is soluble starch (3.17%), wheat bran (0.79%), KH2PO4 (0.1%), MgSO4·7H2O (0.05%), and VB1 (10 mg/100 mL). The production was increased by 253% to 183.46 mg/L from 51.98 mg before optimization.e