In this study, the fruiting body of Cordyceps militaris was used to validate its α-glucosidase inhibitory activity in vitro. Using maltose as the reaction substrate and acarbose as a positive control, in vitro enzymatic reactions were carried out to validate the α-glucosidase inhibitory activity of polysaccharides (with different purities) from Cordyceps militaris, which were purified by water extraction, decolorization, alcohol precipitation, deproteinization, and column chromatography. The results of stepwise enzymatic reaction showed that the functional component with α-glucosidase inhibitory activity in Cordyceps militaris was polysaccharide, and the inhibitory rate gradually increased with increasing purity of the polysaccharide. The results of further enzymatic reactions using Cordyceps militaris polysaccharide purified by column chromatography showed that the rate of α- glucosidase inhibition increased with increasing concentration of polysaccharide, which was demonstrated that the inhibitory activity of Cordyceps militaris polysaccharide was dose-dependent within a certain concentration range. The half-maximal inhibitory concentration (IC50) value of purified polysaccharide was 4.22 mg/mL, showing a good hypoglycemic function. This study provides a theoretical basis for the future development of hypoglycemic drugs and health products using Cordyceps.