Objective: To explore the potential of serum disease-specific immunoglobulin G (DSIgG) glycosylation as a biomarker for the diagnosis of nonproliferative diabetic retinopathy (NPDR). Methods: A total of 387 consecutive diabetic patients presenting in an eye clinic without proliferative diabetic retinopathy (DR) were included and divided into those with nondiabetic retinopathy (NDR) (n = 181) and NPDR (n = 206) groups. Serum was collected from all patients for DSIgG separation. The enriched glycopeptides of the tryptic digests of DSIgG were detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients were randomly divided into discovery and validation sets (1:1). The differences in glycopeptide ratios between the groups were compared by using Student's t-test or the Mann-Whitney U test. The predictive ability of the model was assessed using the area under the receiver operating characteristic curve (AUC). Results: DSIgG1 G1FN/G0FN, G2N/G2, G2FN/G2N and DSIgG2 G1F/G0F, G1FN/G0FN, G2N/G1N, G2S/G2 were significantly different between NDR and NPDR patients (p < 0.05) in both the discovery and validation sets. The prediction model that was built comprising the seven glycopeptide ratios showed good NPDR prediction performance with an AUC of 0.85 in the discovery set and 0.87 in the validation set. Conclusion: DSIgG Fc N-glycosylation ratios were associated with NPDR and can be used as potential biomarkers for the early diagnosis of DR.