In this study, a technique that combines supercritical fluid extraction with ethanol extraction was employed to effectively fractionate egg yolk extracts into non-polar and polar lipids. By utilizing feature based molecular networking (FBMN) and MS-DIAL database search, 35 glycerolipids were identified in the supercritical extract (SE), and 70 glycerophospholipids in the ethanol extract (EE). Following the identification of lipids, a heatmap was generated using R software to visualize the abundance of lipids across both fractions. The results indicated that glycerolipids in SE primarily included C18:1, C16:0, and C18:2 fatty acids. In contrast, the glycerophospholipids present in EE exhibited a significant proportion of polyunsaturated fatty acids, notably docosahexaenoic acid (DHA, C22:6) and arachidonic acid (C20:4). Further analysis using GC-MS revealed that the combined content of DHA and arachidonic acid in EE exceeded 10 %. Moreover, EE exhibited potent anti-inflammatory activities, whereas SE showed significant analgesic effects in zebrafish model.