The aim of this study was to screen the biomarkers of diabetic limb arterial stenosis. Fasting blood samples of 40 patients with diabetic limb arterial stenosis (experimental group), 40 diabetes patients (diabetic control group), and 40 healthy individuals (healthy control group) were collected. Protein-chip assay analysis and ELISA were used to detect tumor necrosis factor α, interleukin-6, endothelin-1, calcitonin gene-related peptide and high-sensitivity C-reactive protein in the three groups. Protein-chip array analysis and ELISA found consistent results that endothelin-1, tumor necrosis factor α, interleukin-6 and high-sensitivity C-reactive protein in the experimental group were significantly up-regulated while the expression of calcitonin gene-related peptide was down-regulated compared with the healthy control group (P < 0.01). When compared with the diabetic control group, only markedly increased calcitonin gene-related peptide and interleukin-6 were observed in the experimental group (P < 0.01). The study suggests that high-throughput protein-chip may be a reliable method to screen biomarkers of diabetic limb arterial stenosis. Calcitonin gene-related peptide and interleukin-6 might be promising biomarkers for diabetic limb arterial stenosis.