Abstract: Objective: To optimize the extraction method of total flavonoids from Peony purpurea seeds with microwave-assisted deep eutectic solvent, and to investigate its antioxidant activity in vitro. Methods: Microwave-assisted technology using deep eutectic solvent as extraction solvent was used to extract total flavonoids from Paeonia rockii seeds. Single factorial experiments and response surface methodology optimized the extraction process. The in vitro antioxidant activity of the total flavonoids was evaluated in terms of the rate of scavenging of DPPH radicals and the rate of scavenging of hydroxyl radicals. Results: Under the deep eutectic solvent system with chloride as hydrogen bond acceptor and urea as hydrogen bond donor, the deep eutectic solvent system with a molar ratio of 1:3 and a water content of 41.80% (V/V) was optimal. The ratio of material to liquid was 1:19 g/mL, the power of the microwave was 110 W and the time of the microwave was 3.00 min. The total flavonoid yield from Paeonia rockii seeds was 1.76 mg/g. When the total flavonoid concentration of the extract was 80 μg/mL, the scavenging rate of DPPH reached 82.00%. And the scavenging rate of hydroxyl radicals was 42.10% when the total flavonoid concentration of the extract was up to 80 μg/mL. Conclusion: Microwave-assisted deep eutectic solvent extraction of total flavonoids from Paeonia rockii seeds has high content, antioxidant activity and DPPH free radical scavenging rate of more than 80%.