Although phosphatidylethanolamine (PE) is an important functional phospholipid, there have been very few reports on its antioxidant activity and corresponding molecular composition. Crude PE was extracted from egg yolk with various solvents, and response surface modeling was carried out to determine the optimum extraction conditions for PE, under which the PE content in extracts reached 58.94 µg/mL. The crude PE was purified using silica gel-based column chromatography. High-purity PE (98%), identified by high-performance liquid chromatography-evaporative lightscattering detector, was obtained using isocratic elution with a mixed solvent (chloroform: methanol: acetic acid = 18:5:1) eluent. PE purified from egg yolk exhibited high radical-scavenging activity, determined by electron paramagnetic resonance (EPR). The result was attributed to the high unsaturated fatty acids (83.10%) content in egg yolk PE, and the unsaturated fatty acids were identified as PE-16:0/18:1 9 , PE-16:0/18:2 9,12 , 8,11,14 , PE-18:0/18:1 9 , PE-18:0/18:2 9,12 ,8,11,14 by MALDI-TOF MS combined with gas chromatography mass spectrometry.Practical Application: In this work, an attempt has been made to explore the antioxidant activity of PE that extracted and purified from egg yolk and its corresponding molecular composition. Owing to its plentiful unsaturated fatty acids (83.10%), purified PE from egg yolk exhibited a high radical-scavenging activity that indicated that egg-yolk PE had a strong antioxidant activity, and it might exert possible beneficial effects on the human health.