Omega-7 (n-7) phospholipids were bioactive substances in marine animals. In this study, a fast lipidomics phenotyping approach for real-time in situ screening of n-7 phospholipids in five kinds of economic seafood, salmon, prawn, bluefin tuna, hairtail, and butterfish, was established using iKnife rapid evaporative ionization mass spectrometry (REIMS). The n-7 phospholipids were structurally characterized and quantitatively analyzed, and the profiles were statistically analyzed by multivariate recognition analysis. It indicated that the difference of n-7 phospholipids in seafood samples was significant (p < 0.05), with R2(cum) and Q2(cum) values of >0.9. The proportion of n-7 phospholipids in salmon was the highest (20.43%), followed by bluefin tuna, prawn, hairtail, and butterfish. The ions of m/z 742.54 (PC 16:1-18:1), 768.55 (PC 16:1-20:2), 697.48 (PE 16:1-18:1), and 699.48 (PE 16:1-18:0) were the main n-7 phospholipids. The effectiveness of iKnife REIMS was further verified by hydrophilic interaction chromatography mass spectrometry and gas chromatography. The results demonstrated that proposed iKnife REIMS was an excellent technique for front-line screening of n-7 phospholipids in a large variety of marine biological resources.