Lycium ruthenicum Murr. (LRM, Solanaceae), also called "black wolfberry" in China, is extremely tolerant drought. Although black wolfberry has seeds that are rich in oil, being considered as a potential alternative for edible oil production, little information has been found regarding its characterization. The present study evaluated the fatty acid profile of the seed oil and its phytosterol contents. Moreover, its antioxidant potential and its inhibition activities on pancreatic lipase, and cholesterol esterase (CEase) were also determined. The key fatty acids of studied oil were linoleic (74.58%), oleic (11.82%), γ-linolenic (6.60%), palmitic (4.88%), and stearic (1.40%) acids, which together comprised 99.28% of the total fatty acids. Phytosterols including 24-methylenecholesterol, campesterol, stigmasterol, dihydrolanosterol, 24-methyldesmosterol, β-sitosterol, Δ5 -avenasterol, cycloartenol, and Δ7- avenasterol were identified for the 1st time. The inhibition values (IC50 ) for the DPPH and ABTS radical scavenging capacities of the oil were 7.86 and 15.49 mg/mL, respectively. Additionally, LRM seed oil was also demonstrated to possess strong pancreatic lipase, and CEase inhibition activities with IC50 values of 12.38 and 2.63 mg/mL, respectively. The mechanism study indicated that the inhibitory manner of the oil on CEase belonged to uncompetitive inhibition of reversible inhibition. The results derived from above studies suggest that LRM seed oil could be a potential source of high value phytochemicals for developing novel functional food.