Natural products with high antioxidant activity have attracted great attention in recent years. A water extraction and alcohol precipitation method were used to extract polysaccharides from the Flammulina velutipes stem-base (FVSP). The series of samples including FVSP30, FVSP45, FVSP60, and FVSP75 were isolated by different concentrations of ethanol. The crude polysaccharides were purified by freeze-thaw; deproteinization; DEAE cellulose-52 chromatography; Sephadex G-100 size-exclusion chromatography; and another series of samples were found including FVSP-1, FVSP-2, and FVSP-3. Through 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical, and superoxide anion scavenging experiments, the effects of ethanol concentration during isolation and the degree of purification on the in vitro antioxidant properties of the FVSPs were analyzed. The results showed that the polysaccharides isolated by different ethanol concentrations exhibited strong antioxidant activity. With increasing ethanol concentration in the isolation process, the antioxidant activity was progressively enhanced. In the experimental concentration range, FVSP75 showed the strongest antioxidant activity at a concentration of 1.0 mg/mL. The FVSPs of different purities exhibited excellent antioxidant activity. Within the experimental range, as the degree of purification of the polysaccharides increased, the antioxidant activity decreased accordingly. FVSP-1, with low purity, exhibited the highest antioxidant activity at a concentration of 1.0 mg/mL. These results should provide a foundation for the use of FVSPs as a nutritional functional food or food additive and provide a theoretical basis for the efficient use of FVSPs.