Abstract: In this study, univariate and Box-Behnken response surface experiments were used to optimize the polyphenol extraction process of Rhodiola rosea. Then, a α-glucosidase inhibition system was constructed in vitro to study the inhibition activity of Rhodiola rosea polyphenols. At the same time, the type of inhibition was judged by enzyme inhibition kinetics. The univariate and response surface results showed that the optimal extraction process conditions were: Ethanol concentration of 71%, feed-to-liquid ratio 1:40, ultrasonic power 320 W, ultrasonic temperature 55 °C. Under this condition, the yield of Rhodiola rosea polyphenol extraction could reach 11.45%. Rhodiola rosea polyphenols had certain inhibitory activities on α-glucosidase, and the inhibition ability was quantitatively effective. Half inhibition concentration (IC₅₀) was 2.83 mg/mL, lower than the positive control acarbose (3.36 mg/mL of IC₅₀). When Rhodiola rosea polyphenol concentration was 50 mg/mL, the inhibition rate could reach 97.86%. Enzyme inhibitory kinetic studies showed that Rhodiola rosea polyphenols were reversible hybrid inhibitory types of α-glucosidase. As the concentration of the inhibitor increased, the maximum reaction rate V_max decreased and the Michael's constant K_m increased, which belonged to the competitive and non-competitive mixed types. This optimization test was effective and feasible, and the extracted Rhodiola rosea polyphenols had good inhibitory activity against α-glucosidase in vitro.