Abstract: To enhance the exopolysaccharide production from Antrodia cinnamomea, condition of submerged fermentation was optimized using a statistical method in this study. Eleven nitrogen sources and five carbon sources were screened for their effect on the production of exopolysaccharide. Plackett-Burman experiments based on one-factor experiments were designed to determine the significant effect factors. Response surface optimization experiments with four factors and three levels based on Plackett-Burman experiments were carried out to determine the optimal fermentation conditions. The optimized conditions were as follows:glucose 60 g/L, yeast extract 16 g/L, vitamin B₁ 1 g/L, KH₂PO₄ 0.75 g/L, MgSO₄·7H₂O 0.77 g/L, initial pH5.0, loading volume 120 mL/500 mL, inoculation of 18%, 27℃, 10 d, 100 r/min. The optimized exopolysaccharide yield was 1.23 g/L, which had increase 71% than previous study. The optimized polysaccharide production had greatly increased, which could provide reference for further industrial production.