Diabetic retinopathy (DR) is a typical microvascular complication of diabetes mellitus (DM) and it remains one of the leading causes of vision loss worldwide. Studies postulated that a distinct metabolic signature of DR exists and can be resolved from that of diabetes alone. Serum Semaphorin3A (Sema3A) levels have also been found to be correlated with the phenotypes of diabetic retinopathy. We aimed to analyze and identify serum metabolites and serum Sema3A levels that could be useful biomarkers of DR progression. This cross-sectional study included 45 type 2 diabetes (T2D) patients. Diabetic patients were divided into three groups based on the status of their complications: non-DR (NDR, n=15), non-proliferative DR (NPDR, n=15), and proliferative DR (PDR, n=15) groups. Serum metabolomic profiles of these patients were determined by using high-performance liquid chromatography-mass spectrometry (HPLC-MS), and serum Sema3A levels measured by ELISA. Metabolomics analysis revealed a set of metabolites that were altered in the serum of PDR patients as compared with NPDR and NDR groups. Among these metabolites total asymmetric dimethylarginine (ADMA) and Kynurenine were potential predictors of PDR patients. Significantly higher serum levels of Sema3A in PDR patients as compared with NPDR and NDR groups (p<0.001), their serum levels were positively correlated with the central macular thickness (r= 0.952, p<0.001) and negatively correlated with the superficial macular density (r=-0.952, p<0.001). In conclusion, the metabolite signatures identified in this study and serum Sema3A levels could be proposed as biomarkers for DR development and progression in T2D patients. However, Sema3A was superior to metabolomics in the prediction of the severity of DR.