Peony seed were extracted by ultrasonic-assisted extraction to produce peony seed oil. The influence of extraction reagent, time, temperature, ultrasound power and liquid-solid ratio on the peony seed oil extraction rate were studied by single factor and orthogonal test. The optimum process conditions of ultrasonic-assisted extraction of peony seed oil was obtained and the composition of peony seed oil fatty acid was analyzed by Gas chromatography. The results showed that the best extraction conditions of ultrasonic-assisted extraction of peony seed oil were: extraction reagent N-hexnane, extraction time 50min, extraction temperature 50 C, ultrasound power 140W, and liquid-solid ratio 1∶15 (g∶mL). Under the above conditions, the peony seed oil extraction yield was (26.11±0.01)%. Gas chromatography analysis showed that the main composition of peony seed oil is palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. Unsaturated fatty acid content was as high as (92.35±0.51) %, linoleic acid and linolenic acid content were (28.80±0.24)% and (41.13±0.09)%, respectively. Peony seed oil is a high nutritional edible oils.