Four extraction techniques including enzyme-assisted supercritical carbon dioxide extraction (ESC-CO2), supercritical carbon dioxide extraction (SC-CO2), ultrasound-assisted extraction (UAE) and Soxhlet extraction (SE) were evaluated to efficiently extract the oil from Decaisnea insignis seeds in this study. Lipid profiling by GC-MS confirmed seven fatty acids and six phytosterols in all oils obtained, in which palmitoleic acid (from 475.4 ± 12.0 by SE to 604.9 ± 26.1 mg/g oil by ESC-CO2) and β-sitosterol (from 35.6 ± 1.2 by SE to 60.9 ± 2.3 mg cholestanol equivalent/100 g oil by ESC-CO2) predominated, respectively. Furthermore, the oil yielded by ESC-CO2 displayed higher polyphenol content and stronger DPPH and ABTS radical scavenging capacities. These results suggested that D. insignis seed oil is considered an alternative source of palmitoleic acid, and ESC-CO2 could be explored as an environmentally friendly technology for extracting plant oil.