The invention relates to the field of aquatic organisms and analysis chemistry, in particular to a method for separating and authenticating the components of fatty acid in the body of a soft-shelled turtle by combining GC-MS (gas chromatography-mass spectrometry) with a retention index. Fatty acid methyl esters are prepared by separating the adipose tissue of the soft-shelled turtle, and extracting and deriving the fatty acid from the separated adipose tissue. The method for separating and authenticating the fatty acid comprises the following steps: dissolving fatty acid methyl esters of the soft-shelled turtle in hexane and analyzing by using GC-MS to fulfill the aim of separating; calculating the retention index of fatty acid methyl esters according to the retention time of different fatty acid methyl esters; searching a mass spectrometry library, and meanwhile, carrying out nature determination by combining the retention index, and calculating the relative content of each fatty acid methyl ester by an area normalization method so as to fulfill the aim of analyzing the components of fatty acid of the soft-shell turtle accurately and quickly. The method has the advantages that the components of fatty acid in the soft-shell turtle can be authenticated accurately by fully utilizing the method of searching the mass spectrometry library and nature determination by combining the retention index; particularly, the operation is simple and convenient, and the method has the advantage that the result is accurate compared with the method of searching the mass spectrometry library.