Abstract: Soluble β-glucan from mushroom source had good biological activity, in order to develop and utilize β-glucan in Cordyceps chrysanthemi, this study used Cordyceps chrysanthemi substrate as raw material, and was purified by proteolytic enzyme digestion, adsorption of macroporous resin, and chromatography of DEAE ion-exchange columns, and the decolorization and β-glucan retention were weighted and scored to feel out the establishment of the optimal purification process conditions. The obtained β-glucan was structurally analyzed and its in vitro hypoglycemic activity was investigated. The results showed that the macroporous resin HPD-750 had the best adsorption effect, and the optimal conditions for adsorption were as follows: up-sample flow rate of 5 mL/min, up-sample concentration of 4 mg/mL, up-sample volume of 400 mL, and mass of the resin of 40 g. Two β-glucan fractions, CMBG-1 and CMBG-2, with relative molecular weights of 13.57×10⁴ Da and 14.36×10⁴ Da, respectively, were isolated using a DEAE-FF cellulose exchange column. Both consisted of three monosaccharides, Gal, Glc, and Ara, with molar ratios of 1.28:5.27:1.01 and 0.71:4.43:0.80, respectively. The Congo red analysis showed that CMBG-1 has a triple-helix structure, while CMBG-2 does not have a triple-helix structure, and the inhibition rates of both of them on α-amylase and α-glucosidase were positively correlated with the concentrations of the samples, and the IC₅₀s of their inhibition on α-amylase were 2.68 and 4.32 mg/mL, and those on α-glucosidase were 2.37 and 3.01 mg/mL, respectively. The hypoglycemic activity of CMBG-1, which possessed a triple-helix structure, was significantly higher than that of CMBG-2, which did not possess a triple-helix structure, and it had good prospects for the development of functional foods.