The optimal fermentation conditions and medium for the production of bioactive polysaccharides from the mycelium of Cordyceps sinensis fungus UM01 were investigated by using orthogonal design and high performance size exclusion chromatography coupled with multi-angel laser light scattering and refractive index detector (HPSEC-MALLS-RID). Results showed that the optimal temperature, initial pH, rotation speed, medium capacity (ratio of medium volume to the volume of flask bottle) and inoculums volume for the mycelium growth were 15°C, pH 6.0, 150rpm, 2/5 (v/v), and 3% (v/v), respectively. Furthermore, bioactive polysaccharides from the mycelium of C. sinensis fungus UM01 were determined as polysaccharide fractions with the molecular weight above 10kDa. The optimal fermentation medium was determined as a composition of glucose 30.0g/L, sucrose 30.0g/L, KH2PO4 1.0g/L, CaCl2 0.5g/L, yeast extract 3.0g/L, and MgCl2 0.1g/L according to the maximum amount of the bioactive polysaccharides (486.16±19.60mg/L) measured by HPSEC-MALLS/RID. Results are helpful to establish an efficient and controllable fermentation process for the industrial production of bioactive polysaccharides from C. sinensis UM01, and beneficial to develop a unique health and functional product in future.