Metabolomics analysis of three Saccharopolyspora spinosa strains (wild type strain WT, ultraviolet mutant strain WH124, and metabolic engineering strain LU104) with different spinosad producing levels was performed by liquid chromatograph coupled to mass spectrometry (LC-MS). The metabolite profiles were subjected to hierarchal clustering analysis (HCA) and principal component analysis (PCA). The results of HCA on a heat map revealed that the large numbers of primary metabolism detected were more abundant in WH124 and less abundant in LU104 during the early fermentation stage as compared to the WT strain. PCA separated the three strains clearly and suggested nine metabolites that contributed predominantly to the separation. These biomarkers were associated with central carbon metabolism (succinic acid, α-ketoglutarate, acetyl-CoA, and ATP), amino acid metabolism (glutamate, glutamine, and valine), and secondary metabolism (pseudoaglycone), etc. These findings provide insight into the metabolomic characteristics of the two high-yield strains and for further regulation of spinosad production.