Abstract: To determine the optimal extraction conditions, chemical composition, and in vitro biological activity of Pueraria polyphenols, Pueraria was used as the raw material. The extraction procedure was optimized using response surface methodology (RSM). The chemical structures of purified Pueraria polyphenols (G₄) were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and the antioxidant and hypoglycemic activities before and after purification were compared. Results showed that the optimal extraction conditions for Pueraria polyphenols were an ultrasound duration of 43 min, a liquid-to-material ratio of 28:1 mL/g, an ultrasound temperature of 69 ℃, and an ultrasound power of 250 W, achieving a polyphenol yield of 6.184%±0.037%. After purification, the α-glucosidase inhibitory rate of G₄ was significantly increased (P<0.05), and the scavenging activities against DPPH and ABTS⁺ radicals were enhanced by 11.15% and 27.69%, respectively. Structural identification revealed that G₄ comprised 12 phenolic compounds, including daidzin, genistin, daidzein, and puerarin-4′-O-β-D-glucoside. Cellular experiments demonstrated that low, medium, and high concentrations of G₄ increased glucose consumption by 4.12%, 9.35%, and 21.54%, respectively, compared with the model group. Glycogen content was elevated by 32.12%, 44.17%, and 57.83%, respectively, and activities of hexokinase（HK）and pyruvate kinase（PK）were significantly enhanced (P<0.05). These findings suggest that G₄ effectively ameliorates insulin resistance in HepG2 cells, exhibiting excellent hypoglycemic activity. In conclusion, this study provides a theoretical basis for the efficient utilization of Pueraria resources and the development of functional foods.