For full utilization of the fishery wastes, the aim of this study was to establish a method for extracting the phospholipids and fish oil simultaneously from wastes, followed by the analysis of phospholipid and fatty acid composition on the basis of multi-dimensional tandem mass spectrometry and gas chromatography. Phospholipids and fish oil were extracted simultaneously by modified Folch method and precipitation method with acetone. The extracting efficiency of five different extracts was compared, and the mixture of CH₂Cl₂-MeOH (1∶2, V/V) performed best, whose extracting rates of phospholipids and fish oil were 0.97%和28.05%, respectively. Afterwards, the obtained fish oil was esterified and analyzed by gas chromatography, and the obtained phospholipids were injected directly by a syringe pump and identified and quantified by tandem mass spectrometry in the precursor-ion scan (PreIS) and neutral-loss scan (NLS) modes. The results show that fish oil contains 23 kinds of fatty acids, including oleic acid, linoleic acid, palmitic acid, etc. The content of unsaturated fatty acid is 75.97%. There are four classes (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine) and 69 kinds of phospholipid molecules were detected in the phospholipid extract. Phospholipids with at least one eicosapentaenoic acyl/docosahexaenoic acyl chain were frequently found, such as 16:0/20:5, 16:0/22:6, 18:1/22:6, 18:0/22:6, 20:0/22:6, etc. In conclusion, this method can efficiently extract phospholipids and fish oil simultaneously. It is simple, fast, and friendly to the environment.