Serum cystatin C (CysC) has been identified as a possible potential biomarker in a variety of diabetic complications, including diabetic peripheral neuropathy and peripheral artery disease. We aimed to examine the association between CysC and diabetic foot ulceration (DFU) in patients with type 2 diabetes (T2D). 411 patients with T2D were enrolled in this cross-sectional study at a university hospital. Clinical manifestations and biochemical parameters were compared between DFU group and non-DFU group. The association between serum CysC and DFU was explored by binary logistic regression analysis. The cut point of CysC for DFU was also evaluated by receiver operating characteristic (ROC) curve. The prevalence of coronary artery disease, diabetic nephropathy (DN), and DFU dramatically increased with CysC (P < 0.01) in CysC quartiles. Multivariate logistic regression analysis indicated that the significant risk factors for DFU were serum CysC, coronary artery disease, hypertension, insulin use, the differences between supine and sitting TcPO2, and hypertension. ROC curve analysis revealed that the cut point of CysC for DFU was 0.735 mg/L. Serum CysC levels correlated with DFU and severity of tissue loss. Our study results indicated that serum CysC was associated with a high prevalence of DFU in Chinese T2D subjects.