An ethanol-assisted aqueous enzymatic extraction was performed for peony seed oil (content of 30%). This method included cooking pretreatment, pectinase hydrolysis, and aqueous ethanol extraction, and the corresponding variables in each step were investigated. The changes in viscosity and dextrose equivalent values of the reaction medium as a function of changing enzymatic hydrolysis time were compared to the oil yield. The microstructures of peony seeds were analyzed using confocal laser scanning microscopy to understand the process of oil release as a result of cooking and grinding. The highest oil yield of 92.06% was obtained when peony seeds were cooked in deionized water with a solid-liquid ratio of 1:5 (w/v) at 110 C for 1 hour, ground to 31.29 μm particle size, treated with 0.15% (w/w) pectinase (temperature 50 C, pH 4.5, time 1 hour), and then extracted with 30% (v/v) aqueous ethanol (temperature 60 C, pH 9.0, time 1 hour). After processing with pectinase followed by ethanol extraction, the residual oil content in water and sediment phase decreased to 5% and 3%, respectively. The quality of the oil obtained by ethanol-assisted aqueous enzymatic extraction was good, complying with the Chinese standard.Deshelled peony seeds were added to deionized water or 0.05 M citric acid solution at a solid-liquid ratio of 1:5 596 J Am Oil Chem Soc J Am Oil Chem Soc (2019) 96: 595-606 Fig. 6 Trends of viscosity and DE value during the pectinase hydrolysis. Other conditions: deionized water cooking (1:5 g mL −1 , 110 C, 1 hour), solid-liquid ratio 1:7 (g mL −1 ). Pectinase hydrolysis: pectinase dosage 0.15% (v/v), temperature 50 C, and pH 4.5 601 J Am Oil Chem Soc J Am Oil Chem Soc (2019) 96: 595-606