Background We hypothesized that specific amino acids or acylcarnitines would have benefits for the differential diagnosis of diabetes. Thus, a targeted metabolomics for amino acids and acylcarnitines in patients with diabetes and its complications was carried out. Methods A cohort of 54 normal individuals and 156 patients with type 2 diabetes mellitus and/or diabetic complications enrolled from the First Affiliated Hospital of Jinzhou Medical University was studied. The subjects were divided into five main groups: normal individuals, impaired fasting glucose, overt diabetes, diabetic microvascular complications, and diabetic peripheral vascular disease. The technique of tandem mass spectrometry was applied to obtain the plasma metabolite profiles. Metabolomics multivariate statistics were applied for the metabolic data analysis and the differential metabolites determination. Results A total of 10 cross-comparisons within diabetes and its complications were designed to explore the differential metabolites. The results demonstrated that eight comparisons existed and yielded significant metabolic differences. A total number of 24 differential metabolites were determined from six selected comparisons, including up-regulated amino acids, down-regulated medium-chain and long-chain acylcarnitines. Altered differential metabolites provided six panels of biomarkers, which were helpful in distinguishing diabetic patients. Conclusion Our results demonstrated that the biomarker panels consisted of specific amino acids and acylcarnitines which could reflect the metabolic variations among the different stages of diabetes and might be useful for the differential diagnosis of prediabetes, overt diabetes and diabetic complications.