Objective: To explore the relationship between serum cystatin C (Cys-C), retinol-binding protein (RBP), urine microalbumin-to-creatinine ratio (ACR), and disease severity in patients with type 2 diabetic nephropathy (T2DN). Methods: A total of 103 patients with T2DN were enrolled as the T2DN group. According to the excretion rate of urinary albumin, they were divided into the early stage group (n=36), clinical stage group (n=55), and uremia group (n=12). A total of 105 patients with type 2 diabetes mellitus (T2DM) during the same period were enrolled as the T2DM group. The levels of serum Cys-C, RBP, and ACR in the T2DN group and T2DM group were detected and compared, and their relationship with disease severity was analyzed. The diagnostic efficiency of Cys-C, RBP, ACR, and combined detection for the severity of T2DN was analyzed by ROC curves. Results: The levels of serum Cys-C, RBP, and ACR in the T2DN group were higher than those in the T2DM group (P<0.05). The levels of Cys-C, RBP, and ACR in the clinical stage group and uremia group were higher than those in the early stage group (P<0.05). Serum Cys-C, RBP, and ACR were positively correlated with disease severity (P<0.05). The AUC of Cys-C combined with RBP and ACR in the diagnosis of T2DN in the clinical stage and uremia stage was 0.934, greater than that of a single index (P<0.05). Conclusion: The changes of Cys-C, RBP, and ACR are positively correlated with the severity of T2DN, and combined detection of the three indexes has higher diagnostic efficiency.