In order to improve the utilization rate of tuna by-products， this study was aimed to establish a method for extracting the phospholipids and fish oil simultaneously from wastes， followed by the analysis of fatty acid and phospholipids composition on the basis of multi-dimensional tandem mass spectrometry and gas chromatography. The 1，2-dichloroethane system combined with acetone precipitation was established to extract phospholipids and fish oil from tuna viscera. After the optimization of this method and the comparison between this method and other traditional methods， the best solvent system was shown to be 1，2-dichloroethane-methanol （1 ∶ 2， V/V）， whose yields of phospholipids and fish oil were 0.994% and 17.306%， respectively. Afterwards， the obtained fish oil was esterified and analyzed by gas chromatography， and the obtained phospholipids were identified and quantified by liquid chromatography/mass spectrometry in the negative-ion full scan modes. The results show that fish oil mainly contains 18 kinds of fatty acids such as DHA， palmitic acid， oleic acid， EPA， et al， among which polyunsaturated fatty acids account for 36.678%. A total of 16 kinds of phosphatidylcholines， 14 kinds of phosphatidylethanolamines， 11 kinds of phosphatidylinositols and 12 kinds of phosphatidylserines were detected in phospholipid samples， and a variety of phospholipids containing omega-3 polyunsaturated fatty acid chains were detected. As a conclusion， this method can not only extract phospholipids and fish oil in the tuna by-products simultaneously， but also be safer and more suitable for food processing industry. Meanwhile， it can develop and utilize tuna by-products reasonably and achieve the purpose of protecting the environment efficiently.