Zizyphi Spinosae Semen (ZSS) is an herbal medicine with a variety of biological activities. In this study, the oil fraction of ZSS was extracted using an ultrasonic assisted extraction (UAE) method and the value of this underutilized byproduct was explored. The effects of operational parameters of the UAE process (extraction temperature, extraction time, liquid-to-solid ration, and ultrasonic power) on the extraction efficiency were evaluated based on the yield of vitamin E and the squalene, two important compounds in ZSS oil with antioxidant bioactivities. A central composite design was used to optimize these operational parameters. The optimized UAE process resulted in a vitamin E yield of 29.58 ± 0.19 mg/100 g ZSS and a squalene yield of 820.49 ± 3.66 µg/100 g ZSS, which were much higher than those obtained through the conventional heating reflux extraction (HRE) (i.e., vitamin E yield of 18.67 mg/100 g ZSS, and squalene yield of 627.25 µg/100 g ZSS). The ZSS oil contained oleic acid and linoleic acid as the main fatty acid. In addition, the UAE-derived ZSS oil exhibited stronger activities of scavenging ABTs, superoxide anions and hydroxyl radicals, compared to the ZSS oil extracted from HRE. Collectively, this work demonstrated that UAE was an effective method to extract ZSS oil with high yield. The ZSS oil can be used as a potential an antioxidant agent in food and pharmaceutical industries.