Abstract Response surface methodology (RSM) was used to optimize ultrasound-assisted aqueous enzymatic extraction (UAAEE) conditions for perilla seed oil. Under the optimum conditions—which were a liquid-to-solid ratio of 4.4:1, a hydrolysis time of 2.66 h, a hydrolysis temperature of 50.87 °C and an ultrasound treatment time of 24.74 min—an oil yield of 31.34% was obtained. Comparisons of the physicochemical characteristics of oil obtained using UAAEE with those of oil obtained by solvent extraction (SE oil) and cold pressing extraction (CPE oil) revealed similar refractive indices and saponification values, but UAAEE oil had a higher iodine value and better stability against oxidation, with a low peroxide value. UAAEE oil contained higher levels of beneficial α-linolenic acid and phenolics than SE oil or CPE oil, and it also had superior efficiency in scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.