In this study， gas chromatography (GC) and high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry(HPLC-QTRAP-MS/MS) were used to explore the changes of fatty acids and oxylipins in DHA-enriched egg yolk refrigeration storage period (4 ℃±1 ℃). The results showed that the storage time had an important effect on lipid oxidation in egg yolk. With the increase of storage time， the polyunsaturated fatty acid (PUFA) in egg yolk showed the tendency declining first (from 107.24 mg/g to 93.86 mg/g)， and followed increased (gradually rising to 105.81 mg/g) and then slowly decreased again (to 102.33 mg/g at day 56)， and the ω-6/ω-3 PUFA ratio first decreased and rising up in late. The arachidonic acid (AA) and docosahexaenoic acid (DHA) metabolism were the key pathway of lipid oxidation in DHA-enriched egg yolk， and the 28th day was the key point to distinguish between oxidation and deterioration. During the first 28 days of storage， yolk quality was relatively stable， and the main lipid changes that occurred were AA metabolism to produce 15-HETE and lipid epoxide downregulation. However， after day 28， lipid oxidation began to intensify， and lipid hydroxyl oxides (HETE， HDHA， HODE， HOTrE) and oxyoxides (oxoODE) became the most critical lipid oxidation markers in related metabolic pathways. This study provided a theoretical basis for the identification of yolk oxidation degree and quality control during storage of DHA-enriched eggs.