Abstract: This study used ultrasound-enzyme-assisted deep eutectic solvents to establish a green and highly efficient total flavonoids extraction process from mulberry leaves. On the basis of the results of single-factor experiments, the extraction amount of total flavonoids was used as the response value, and the Box-Behnken response surface method was used to optimize, and the antioxidative ability of total flavonoids from mulberry leaves on DPPH radicals and ABTS⁺ radicals was studied. The results showed that when the choline chloride/fructose/alcohol molar ratio was 1:1:3, water content 30%, liquid-solid ratio 40 mL/g, ultrasonic power 360 W, ultrasonic temperature 40 ℃, ultrasonic time 40 min, dosage of enzyme 4%, the extraction amount of total flavonoids from mulberry leaves reached 46.58 mg/g. When the mass concentration of total flavonoids from mulberry leaves was 0.08 mg/mL, the scavenging rate of DPPH radicals was 98.36%. And when the mass concentration of total flavonoids from mulberry leaves was 0.2 mg/mL, the scavenging rate of ABTS⁺ radicals was 72.12%. Therefore, the ultrasonic-enzyme-assisted deep eutectic solvents method was green and simple to operate, and could effectively improve the extraction yield of total flavonoids from mulberry leaves. This method provided a scientific basis for the development and utilization of mulberry leaf resources.