Metabolomics has become an important tool in distinguishing changes in metabolic pathways and the diagnosis of human disease. Polycystic ovary syndrome (PCOS) is a relatively complicated, heterogeneous endocrine disorder. The etiology and pathogenesis of PCOS remain uncertain. In this study, based on the platform of ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and the method of pattern recognition, a comprehensive metabolomics approach has been applied to explore the changes in metabolic profiling between PCOS patients (n = 20) and controls (n = 15) as well as insulin-resistance (IR) PCOS patients (n = 11) and non-IR PCOS subjects (n = 9) in serum. In total, 36 metabolites were found significantly different between PCOS and controls, and 9 metabolites were discovered significantly different between IR and non-IR PCOS patients. Significant increases in the levels of saturated and unsaturated fatty acids (myristic acid, linoleic acid, 9-/13-HODE, etc.), fatty amides (palmitic amide, oleamide), dehydroepiandrosterone sulfate, L-glutamic acid, azelaic acid, L-glyceric acid, pyroglutamic acid, and decreases in the levels of lysophosphatidylethanolamine, lysophosphatidylcholine, uridine, and L-carnitine were found in PCOS patients compared with controls. In IR PCOS patients, linoleic acid, myristic acid, palmitoleic acid, and vaccenic acid also increased significantly compared with non-IR PCOS patients. All these changed metabolites showed abnormalities of steroid hormone biosynthesis, amino acids and nucleosides metabolism, glutathione metabolism, and lipids and carbohydrates metabolism in PCOS patients. The subgroup IR PCOS patients exhibited greater metabolic deviations than non-IR PCOS patients. These findings may help yield promising insights into the pathogenesis and advance the diagnosis and prevention of PCOS. Graphical Abstract Serum metabolomics signature of polycystic ovary syndrome.