Simple and rapid quantitative determination of fatty acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae.Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes.This study described a gas chromatography-mass spectrometry (GC-MS) method for quantification of seven free fatty acids, nine fatty acid methyl esters, six fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type and genetically engineered cyanobacteria (Synechocystis PCC 6803).Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons.The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated.The results demonstrated that fatty acid-based biofuels can be simultaneously and directly detected without derivation, so rapid and reliable quantitative analysis of fatty acid-based biofuels produced by cyanobacteria can be achieved using the GC-MS method founded in this work.