This study compared the gut microbiota and metabolite profiles of healthy control (HC) and major depressive disorder group (MDD) to identify potential biomarkers that could aid in the diagnosis of major depressive disorder. Gut microbiota composition of 69 patients (42 HC and 27 MDD) was analyzed using 16S rRNA gene sequencing. Urinary metabolite profiles were determined using GC-MS, while plasma metabolite profiles were identified using both GC-MS and UPLC-QTOF-MS. There were no significant differences in overall microbial diversity, but MDD patients showed a higher abundance of [Eubacterium]_eligens_group and Veillonella. A total of 57 plasma metabolites were identified to have statistically significant differences between the two groups. Metabolites associated with neurotransmission and energy metabolism, including serotonin, GABA, citric acid, and betaine, were found to be higher in the HC group compared with the MDD group. MSEA identified glycerophospholipid metabolism as the significantly enriched pathway with the highest enrichment ratio, highlighting the importance of glycerophospholipids in distinguishing MDD from HC. ROC curve analysis demonstrated that plasma metabolomics using UPLC outperformed other profiling, while a combined approach incorporating four datasets achieved the highest predictive accuracy (AUC = 0.88). These findings indicate that integrating gut microbiota and metabolite profiles may provide preliminary insights into MDD classification.