In this study, the recovery of pecan nut kernel oil (PNKO) obtained by mechanical pressing (MP) was compared with that obtained by ultrasonic-assisted aqueous enzymatic extraction (UAAEE). At the same time, contents of substances with proven bioactivity, fatty acid compositions, physicochemical properties and antioxidant activities of PNKO were also assessed. Obviously, the oil yield obtained by UAAEE (71.32%) was higher than that obtained by MP (56.81%). Therefore, Plackett-Burman design (PBD), as well as Box-Behnken design (BBD), further optimized the UAAEE process, and the highest oil yield of 78.83% was acquired under the following conditions: incubation time of 2 h, the mixed enzyme (cell ulase+hemicellulase+pectinase+neutrase, w/w/w/w=1/1/1/1) concentration of 2.9%, liquid-solid ratio of 4 mL/g, pH of 4, particle size of 300 μm, ultrasonic time of 20 min, ultrasonic power of 432 W and reaction temperature of 53℃. The PNKO obtained by the two methods possessed similar fatty acid composition, but that obtained by UAAEE had comparatively low acid value and peroxide value. Moreover, rich content of total phenolics, squalene and phytosterols, as well as strong scavenging activities against DPPH and ABTS + free radicals, were also contained in the PNKO obtained by UAAEE. These results demonstrated that UAAEE was an efficient method to acquire pecan nut kernel oil with excellent quality and high recovery.