Selenium-enriched of Lyophyllum decastes mycelium was cultured in 20L fermenter as raw material in this study. The extraction process of selenium polysaccharide from mycelium of Lyophyllum decastes was optimized and the structure of refined polysaccharides were characterized by IR spectroscopy. One-factors-at-a-time experiments and Box-Behnken experimental design coupled with response surface methodology were carried out to optimize the process parameters of Se-polysaccharides. The results showed that the optimum conditions were: 50 min at 83 ℃, the ratio of solid to liquid 1∶130 (g∶mL), and two times extraction. Under the above conditions, the extraction rate of polysaccharide was 44.62%, which was 0.02% more than predicted, and the selenium content achieved 31.9 μg/g in polysaccharide. After purifying, the refined polysaccharides were characterized by IR spectroscopy. The result showed that the water soluble polysaccharides were pyranopolysaccharide linked by β-glycosidic. The primary structure of water-soluble polyaccharides was not changed, but absorption peaks of pyranring was changed. This further experiment demonstrated that Se participated in the synthesis of Se-polysaccharides during the cultivation of Se-enriched Lyophyllum decastes mycelium.