Abstract: Chestnut pollen, rich in high-value protein, was extensively discarded due to the lack of efficient extraction technologies, limiting its potential applications in functional foods and pharmaceuticals. This study presented the first systematic comparative analysis of three extraction methods—Ultrasound-assisted alkali dissolution and acid precipitation (UADAP), microwave-assisted alkali dissolution and acid precipitation (MADAP), and ammonium sulfate precipitation (ASP)—on the structural attributes and biological activities of chestnut pollen protein. With protein yield as the optimization criterion, the extraction process was systematically optimized through single-factor experiments integrated with response surface methodology. Results showed that all three methods yielded typical proteins with molecular weights of 20~100 kDa, exhibiting characteristic ultraviolet and infrared absorption peaks. Thermogravimetric analysis revealed thermal degradation at approximately 280 ℃, with MADAP-extracted protein demonstrating the highest thermal stability. Antioxidant activity assays indicated that MADAP protein exhibited superior activity in DPPH radical scavenging, hydroxyl radical scavenging, and total antioxidant capacity (T-AOC), with an activity order of MADAP>UADAP>ASP. For hypoglycemic activity, the three chestnut pollen proteins all exhibited favorable inhibitory activities against α-glucosidase and α-amylase. Among them, MADAP showed the optimal inhibitory effect on α-glucosidase, UADAP possessed the strongest inhibitory activity against α-amylase, while ASP displayed the weakest inhibitory capacity toward both digestive enzymes. Response surface methodology optimized MADAP conditions (solid-to-liquid ratio 1:25 g/mL, extraction time 60 min, temperature 60 ℃), achieving a protein yield of 26.41%. In conclusion, the MADAP method effectively preserved protein structural stability while significantly enhancing its antioxidant and hypoglycemic activities, providing an optimal technical approach for the efficient development of chestnut pollen protein.