Characterization of phospholipids (PLs) and phytosterols were determined in Schisandra chinensis (S. chinensis) oil by UPLC-Q/TOF-MSE. The determination process was based on feature fragment information of components generated by the MSE detector. A total of 49 and 39 PLs were identified in S. chinensis oil under negative and positive ion mode, respectively. The classes of PLs included phosphatidic acids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidylinositols (PIs), phosphatidylserines (PSs) and phosphatidylcholines (PCs). The most diverse species of PLs detected were PIs and PCs, accounting for 12 and 14, respectively. The analysis of quantification indicated that PEs and PCs were the most abundant constituents in S. chinensis oil, accounting for 122.85 ± 3.82 and 85.61 ± 2.12 μg/g, respectively. Besides, thirteen kinds of phytosterols were tentatively identified in S. chinensis oil under positive ion mode, among which, conicasterol C was the most abundant component, accounting for 22.02 ± 0.98 μg/g. Brassicasterol, campesterol, secosterol-B and herbasterol were also determined in S. chinensis oil. These results might be meaningful in the quality assessment and function evaluation of S. chinensis oil.