Abstract: To investigate the extraction method of polysaccharide from black corncob and its inhibitory effect on α-glycosidase, black corncobs were used as the raw material in this study, and polysaccharide from black corncob was extracted using ultrasonic-assisted deep eutectic solvent extraction. The yield of polysaccharide and the inhibition rate of polysaccharide from black corncob α-glycosidase were used as indicators to screen for the optimal deep eutectic solvent. Additionally, the content of total sugar, reducing sugar, protein, and uronic acid in polysaccharides from black corncob were determined. The monosaccharide composition, molecular weight distribution, and functional group characteristics of polysaccharide from black corncob were systematically analyzed using ion chromatography, high-performance gel permeation chromatography, and Fourier transform infrared spectroscopy. The inhibition rates of black corncob polysaccharide on α-amylase and α-glucosidase were determined, and the inhibition type was studied in combination with enzymatic reaction kinetics. The results showed that the highest yield of polysaccharide from black corncob prepared by ultrasonic-assisted urea and choline chloride was 5.43%±0.49%. The content of total sugar of polysaccharide from black corncob was 78.0%±2.27%, the content of reducing sugar was 17.2%±0.24%, the content of polysaccharide was 60.8%±0.68%, the content of uronic acid was 3.76%±0.43%, and the content of protein was 4.21%±0.18%. The polysaccharide from black corncob was composed of glucose, galactose, arabinose, xylose, fucose and glucuronic acid. It possesses typical absorption peaks of polysaccharide, a pyran ring structure and α and β glucoside bonds. The weight-average molecular weight was 29487 Da and the number-average molecular weight was 29285 Da. The results of enzymatic reaction kinetics showed that polysaccharide from black corncob showed strong inhibitory activity against α-glucosidase and α-amylase, with IC₅₀ values of 2.49 mg/mL and 6.06 mg/mL, respectively. The inhibition type was reversible inhibition, and the competition mode was high-activity competition. The K_m values were 0.44 mg/mL and 1.746 mg/mL, respectively, and the V_max values were 0.04 and 0.37, respectively. The yield of polysaccharide from black corncob could be significantly improved by ultrasonic-assisted deep eutectic solvent extraction, and the prepared polysaccharide exhibits a strong inhibitory effect on α-glycosidase.