Peony seed oil was obtained by ethanol-assisted aqueous enzymatic extraction after the acid-heat pretreatment on peony seeds. The microstructure of pretreated peony seeds was analyzed by confocal laser scanning microscope. The optimal conditions of this aqueous enzymatic extraction of peony seed oil through the optimization were: finely grounding 8 times (the particle size was 33.62 μm), solid-liquid ratio of 17 (g/mL), the α-amylase enzymolysis at 70 ℃ for 1 h, pH 5.5,dosage 2 mL/100 g·material), and the glucoamylase enzymolysis at 60 ℃ for 1 h, pH 4.5, dosage 3 mL/100 g·material), the ethanol solution extraction at 60 ℃ for 1 h, pH 9.0, the volume fraction of 35%. Under the optimal conditions, the free oil yield reached 90.08%, and the residual oil content in water and sediment phase was 6.60% and 2.78% respectively. The quality indexes of the peony seed oil obtained by the aqueous enzymatic extraction were determined to show that the quality of the crude oil was excellent so that all the indexes reached the standard of the first-grade peony seed oil after simple refining.