To explore the structure and mechanism of hypoglycemic activity in vitro of polysaccharides from Cornus officinalis. Using Cornus officinalis as raw material, the polysaccharides were extracted by ultrasonic water extraction. COP-1S, COP-2S, and COP-3S were obtained by sevage deproteinization, alcohol precipitation, DEAE-52, and Sephadex G-100 column chromatography purification. The hypoglycemic activities in vitro of the three polysaccharide components were evaluated. On this basis, the structures of the polysaccharides were characterized by molecular weight, monosaccharide composition, infrared spectroscopy, etc. The inhibition mechanism of COP-2S on α-glucosidase was explored by enzyme kinetics and fluorescence spectroscopy. The results of hypoglycemic activity showed that the three polysaccharide components had weak inhibitory activity on α-amylase, but significant inhibitory effects on α-glucosidase. The order of inhibitory ability was COP-2>COP-1>COP-3, and the IC₅₀ of COP-2 was 5.18 mg/mL. The average molecular weights of the three polysaccharides were 24.518×10³, 32.392×10³ and 66.417×10³ Da, respectively. The monosaccharide compositions all contained mannose, rhamnose, glucose, xylose and arabinose, but the composition ratios were different. The arabinose composition ratio in COP-1S was relatively high, and both COP-2S and COP-3S contained arabinose and galacturonic acid, but the composition ratio in COP-2S was higher. UV and IR spectra indicated that the three polysaccharides did not contain protein and nucleic acid, and all contained pyran ring structures. COP-2S and COP-3S contained both α and β configurations glycosidic bonds, while COP-1S was of β configuration. Circular dichroism spectroscopy showed that all three polysaccharides increased the β-turn and random coil ratios of α-glucosidase, while decreasing the β-folding ratio, However, only COP-1S increased the α-helix ratio. Enzyme kinetics analysis indicated that the inhibition type of COP-2S on α-glucosidase was mixed competitive inhibition, with an inhibition constant K_i of 0.2248 mg/mL. Fluorescence spectroscopy analysis showed that the quenching effect of COP-2S on α-glucosidase was static quenching, which was a kind of interaction mainly driven by hydrogen bonds and van der Waals forces. This experiment provided a certain theoretical basis for the development of high-value functional foods with hypoglycemic effects from Cornus officinalis.