Abstract: Objective: Optimization of extraction technology of total flavonoids from roots of Cichorium intybus L., and to study its antioxidative activity in vitro. Methods: On the basis of single-factor test, Box-Behnken test design method was used to establish a quadratic regression model with ultrasonic time, liquid-solid ratio, enzymatic hydrolysis time, ultrasonic power and enzyme (cellulase and pectinase) dosage as independent variables, and the total flavonoids yield of chicory roots as the responsive values. The antioxidative activity in vitro of total flavonoids from chicory roots extracted under optimized conditions was evaluated by scavenging ABTS and DPPH free radicals. Results: The optimum extraction conditions of total flavonoids of chicory roots by ultrasound assisted with complex enzyme were as follows: enzyme dosage was 2.2%, liquid-Solid ratio was 37:1 mL/g, enzymatic hydrolysis time was 66 min, ultrasonic power was 59 W, and ultrasonic time was 24 min. Under these conditions, the total flavonoids yield was 5.43±0.12 mg/g. The scavenging rates of DPPH and ABTS radical were 84.45% and 98.18% respectively at the concentration of total flavonoids solution was 0.1 mg/mL, and IC₅₀ were 0.04 and 0.021 mg /mL, respectively. Conclusion: In this study, the extraction process of total flavonoids from chicory roots was optimized by response surface method, and a simulated regression equation of total flavonoids yield was established, which could be used for parameter optimization of extraction process of total flavonoids from chicory roots. The total flavonoids of chicory roots had good antioxidant activity in vitro and could be used as food additives and to develop new antioxidant drugs.