Abstract: Unsaturated fatty acids (UFA) exist widely in organisms, participate in many life activities and play important physiological functions. However, due to the poor functional groups and the existence of a large number of isomers, the structural identification and detailed analysis of UFA face many challenges. Mass spectrometry (MS) analysis has become the main method for the detection of UFA because of its high selectivity, good sensitivity, and the ability to provide molecular mass and fragmentation information. The MS analysis methods for structural characterization of UFA were summarized from the 3 ways of the fat carbon chain analysis, double bonds position analysis and cis-trans isomerism analysis, and the development trend of MS analysis methods was prospected (101 ref. cited).