Abstract: In order to develop a submerged fermentation medium for high-yield extracellular polysaccharides (EPS) of Tremella fuciformis spore, to improve its fermentation yield and achieve large-scale production applications, this study used a single factor method to explore the effects of different sources of carbon and nitrogen sources and nutrient elements on the yield of Tremella polysaccharides. Through the Plackett-Burman and Box-Behnken response surface design to obtain the high-yield EPS fermentation medium and fermentation conditions, and verified the optimization results in a 50 L fermentation tank. The results showed that the fermentation medium of Tremella spores with high production of extracellular polysaccharides was: Glucose 35 g/L, NaCl 0.6 g/L, compound nitrogen source (yeast extracts:corn steep dry powder=1:1) 3.6 g/L, MgSO₄ 0.5 g/L, KH₂PO₄ 1 g/L; Optimal fermentation conditions were: 27 ℃, pH5, fermentation for 6 days, inoculation amount 3%(v/v) and shaker speed 150 r/min. After optimization, the yield of extracellular polysaccharide from Tremella spore was 214.45 mg/100 mL, with 8.56 times of that before optimization. In the 50 L fermentation tank, the polysaccharide yield was 258.78 mg/100 mL with 21.03% higher than the shake flask experiment. This optimization of the fermentation process significantly promoted the production of extracellular polysaccharides from Tremella spore, which would provide a theoretical support for its large-scale industrial production.