Abstract: Diabetic retinopathy (DR), a common microvascular complication, is the leading cause of visual impairment among the working-age population. Late-stage DR can lead to irreversible vision loss, whereas vision impairment can be prevented by controlling blood sugar in a normal level in the early stage of DR. Notably, as early-stage DR is typically asymptomatic, the majority of DR patients are diagnosed at an advanced stage, which causes a great burden to the patients. Therefore, the development of accurate and efficient biomarkers for the early diagnosis of DR is essential for the prevention of DR. In recent decades, a large number of studies have found that serum complement C3 is closely associated with the development of DR. In this study, a method for the detection of complement C3 by mass spectrometry was established by optimizing the enrichment conditions of glycopeptides using C18 materials. It was found that among volunteers with different stages of DR, the level of complement C3 galactose modification showed a tendency to rise first and then go down with the development of DR. The difference in complement C3 glycosylation was used for the diagnosis of different stages of DR, yielding an area under curve (AUC) of up to 0.761.