[Objective] This study aimed to optimize extraction from the leftover of Cordyceps militaris the conditions for polysaccharide culture medium. [Method] Firstly the compositions of C. militaris culture medium were detected, before the cordycepin polysaccharide in medium was extracted using enzymatic hydrolysis. The optimum hydrolytic enzyme was selected through single factor test. Then, the extraction tem- perature, pH, enzyme content and solid-liquid ratio were optimized by response sur- face methodology, and confirmed by mathematical simulation. [Result] Acid hydrolytic enzyme was the optimum for extracting polysaccharides from the leftover of C. mili- taris culture medium. The optimum extraction conditions were as follows: extraction temperature 39.89 ℃, solid-liquid ratio 1:75.78, enzyme content 2.39% and pH 3.12. Under these conditions, the extraction rate of polysaccharides reached 9.96%. [Con- clusion] The study could provide a certain theoretical direction for extracting polysac- charities from the leftover of C. mliltaris culture medium on a large scale.