Background <p>To analyze the potential role of serum and urinary mindin as biomarkers for diabetic retinopathy (DR) severity in patients with type 2 diabetes mellitus (DM).</p> Methods <p>This prospective, cross-sectional study included 101 participants allocated into four groups: DM without DR (<i>n</i> = 26), DM with non-proliferative DR (<i>n</i> = 25), DM with proliferative DR (<i>n</i> = 25), and non-diabetic healthy controls (<i>n</i> = 25). Serum and 24-hour urinary mindin levels were compared among the groups.</p> Results <p>Serum mindin levels were significantly lower in Group 3 than in Group 1 and Group 2 (<i>p</i> &lt; 0.001 and <i>p</i> = 0.001, respectively). Twenty-four-hour urinary mindin levels were significantly higher in the diabetic groups (Groups 1, 2, and 3) compared to the non-diabetic control group (<i>p</i> = 0.046 for Group 1, <i>p</i> &lt; 0.001 for Groups 2 and 3). Correlation analysis revealed positive correlations between 24-hour urinary mindin levels and DR severity, HbA1c, proteinuria, and insulin use. Multivariate logistic regression analysis established HbA1c, urinary mindin level, and proteinuria as independent determinants of DR. Conversely, the serum mindin level was not a significant predictor of DR.</p> Conclusions <p>Twenty-four-hour urinary mindin levels were significantly higher in patients with type 2 DM than in non-diabetic healthy controls. Furthermore, urinary mindin levels increased in correlation with DR severity. In contrast, serum mindin levels were not associated with DR according to multivariate analysis.</p>