BACKGROUND: Major depressive disorder (MDD) is characterized by persistent depressed mood and cognitive symptoms. This study aimed to discover biomarkers for MDD, explore its pathological mechanisms, and examine the associations of the identified biomarkers with clinical and psychological variables. AIM: To discover candidate biomarkers for MDD identification and provide insight into the pathological mechanism of MDD. METHODS: The current study adopted a single-center cross-sectional case-control design. Serum samples were obtained from 100 individuals diagnosed with MDD and 97 healthy controls (HCs) aged between 18 to 60 years. Metabolomics was performed on an Ultimate 3000 UHPLC system coupled with Q-Exactive MS (Thermo Scientific). The online software Metaboanalyst 6.0 was used to process and analyze the acquired raw data of peak intensities from the instrument. RESULTS: The study included 100 MDD patients and 97 HCs. Metabolomic profiling identified 35 significantly different metabolites (, cortisol, sebacic acid, and L-glutamic acid). Receiver operating characteristic curve analysis highlighted 8-HETE, 10-HDoHE, cortisol, 12-HHTrE, and 10-hydroxydecanoic acid as top diagnostic biomarkers for MDD. Significant correlations were found between metabolites (, some lipids, steroids, and amino acids) and clinical and psychological variables. CONCLUSION: Our study reported metabolites (some lipids, steroids, amino acids, carnitines, and alkaloids) responsible for discriminating MDD patients and HCs. This metabolite profile may enable the development of a laboratory-based diagnostic test for MDD. The mechanisms underlying the association between psychological or clinical variables and differential metabolites deserve further exploration.