The extraction process of total flavonoids from Mesona chinensis Benth was optimized, and their hypoglycemic efficacy was evaluated. The optimum extraction conditions were explored using single-factor tests and response surface methodology. The optimum conditions were determined as follows: ethanol concentration, 86%; liquid-solid ratio, 1:18 g/g; ultrasonic extraction time, 36 min; and ultrasonic extraction temperature 66 ℃. Under the above optimum conditions, the predictive extraction rate of total flavonoids from M. chinensis was 2.15%, while the actual extraction rate was 2.17%, with a relative error of 0.93% between the actual and predictive yields. The effects of the four factors on the extraction rate of total flavonoids from M. chinensis were as follows: ultrasonic extraction time>ultrasonic extraction temperature>ethanol concentration>solid-liquid ratio. Additionally, the interaction between ultrasonic extraction time and ultrasonic extraction temperature had a significant (P<0.05) effect on the extraction rate of total flavonoids. The results of in vitro hypoglycemic test showed that the effect of total flavonoids (6.0 mg/g) on the inhibition rate of α-glucosidase activity was 72.18%, whereas the effect of total flavonoids (8.0 mg/g) on the inhibition rate of α-amylase activity was 64.57%. In addition, 10 μg/g of total flavonoids could significantly (P<0.05) increase the glucose consumption of Human Hepatocellular Carcinoma Cell Line cells and promote liver glycogen synthesis (increased by 45.4%). These findings indicated that the total flavonoids from M. chinensis exert a notable auxiliary hypoglycemic effect. The results in this study could provide a theoretical basis for further exploration and potential utilization of the total flavonoids from M. chinensis.