Type 2 diabetes mellitus (T2DM) is a major contributor to peripheral artery disease (PAD), especially in cases that advance to critical limb ischemia (CLI). Accumulating evidence indicates that miRNAs play an important role in the development of PAD and T2DM. Due to the limited value of current diagnostic methods for CLI in T2DM patients, we compared the miRNA expression profiles of Chinese T2DM patients with or without CLI to find out whether distinctive miRNAs could serve as potential diagnostic biomarkers. We statistically identified 7 miRNAs (hsa-miR-200b-3p, hsa-miR-2115-3p, hsa-miR-431-5p, hsa-miR-486-5p, hsa-miR-210-3p, hsa-miR-1264, hsa-miR-323b-5p) which were up-regulated in the CLI group, whereas other 4 miRNAs (hsa-miR-5579-3p, hsa-miR-665, hsa-miR-4285, hsa-miR-500a-3p) were down-regulated. Our validation test suggested a relatively high diagnostic accuracy of serum hsa-miR-323b-5p levels for the detection of CLI in T2DM patients, with a sensitivity of 62.67% and a specificity of 80.65%. The area under the curve (AUC) for miR-323b-5p + confounding risk factors was 0.94 (95% CI: 0.884-0.994, P < 0.001), which was higher than that for miR-323b-5p. Taken together, our results indicate that circulating hsa-miR-323b-5p could be a promising serum biomarker for the diagnosis of critical limb ischemia in type 2 diabetic patients.