Abstract: Shake flask culture was used and the yield of exopolysaccharide（EPS）was taken as main index to ensure the optimum culture conditions for submerged fermentation of Perenniporia fraxinea. The carbon-source， nitrogen-source and cultivation conditions of P. fraxinea in submerged culture were optimized by single factor experiments and multi factor orthogonal experiments respectively. The results showed that the optimal carbon source was surge（40 g/L）and nitrogen source was ammonium nitrate（1 g/L）. In addition，the optimal temperature，initial pH， bottled fluid volume，shaking speed and agitation time for cultivation of P. fraxinea were 27℃， pH 5.5，125 mL，150 r/min，and 9 days. Under the optimal culture conditions，the yield of EPS significantly increased by 55.6% compared with no optimization.