Objective: This study aimed to purify crude polyphenols from wormwood and to study inhibitory activity of its against α-glucosidase. Methods: The adsorption-desorption properties of wormwood polyphenols by seven kinds of macroporous resins were studied. The suitable resin for separating and purifying Wormwood polyphenols was obtained and the purification parameters were optimized. Using different concentration ethanol solution for dynamic elution, the inhibitory activity of different elution fractions on α-glucosidase was investigated. Results: The results indicated that D101 resin was a suitable material for purifying wormwood polyphenols and the macroporous resin reached equilibrium within 120 min. The optimal conditions for purifying wormwood polyphenols were as follows, crude polyphenol 1.5 mg/mL, pH 2.0, flow velocity of 1.5 mL/min, and 60% ethanol was used as the eluent with the elution flow rate of 1.5 mL/min. The purity of polyphenols increased from 21.42% to 69.19% after purification by D101 resin, which was enhanced 3.23 times. Different elution components were obtained by using different concentration ethanol as eluents, respectively. The 60% ethanol elution fraction that contained isoquercitrin, phloridzin and rutin had the best inhibitory effect on α-glucosidase activity. Conclusion: wormwood polyphenols have good development prospects for hypoglycemic effect.