We performed ultrasound-assisted extraction coupled with natural deep eutectic solvents (NADES) to achieve the green and efficient preparation of flavonoid extract from Ampelopsis grossedentata leaves. We then evaluated its antioxidant and antiproliferative activities. A NADES consisting of choline chloride and glucose at a molar ratio of 4:1 with 20% water was determined to be the most suitable solvent. The optimal extraction conditions were: a liquid-to-solid ratio of 30 mL/g, an ultrasonication power of 490 W, and an ultrasonication time of 6.5 min. The actual flavonoid yield was 83.93%, which was close to the predicted yield. Further, 86.75% of the flavonoids were recovered by adding the same volume of phosphate buffer saline (100 mM, pH of 7.0) to the extract solution. Although the chemical antioxidant activities of the flavonoid extract were slightly inferior to those of dihydromyricetin, the flavonoid extract could still effectively inhibit the proliferation of human breast MDA-MB-231 cells by inducing cell apoptosis, retarding the cell cycle, changing the mitochondrial membrane potential and scavenging intracellular reactive oxygen species (ROS). The obtained results can provide a reference in the development of plant-derived functional foods.