Background and Aims: Non-alcoholic fatty liver disease (NAFLD) affects one-quarter of individuals worldwide. Liver biopsy, as the current reliable method for NAFLD evaluation, causes low patient acceptance because of the nature of invasive sampling. Therefore, sensitive non-invasive serum biomarkers are urgently needed. Results: The serum gene ontology (GO) classification and Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed the DEPs enriched in pathways including JAK-STAT and FoxO. GO analysis indicated that serum DEPs were mainly involved in the cellular process, metabolic process, response to stimulus, and biological regulation. Hepatic proteomic KEGG analysis revealed the DEPs were mainly enriched in the PPAR signaling pathway, retinol metabolism, glycine, serine, and threonine metabolism, fatty acid elongation, biosynthesis of unsaturated fatty acids, glutathione metabolism, and steroid hormone biosynthesis. GO analysis revealed that DEPs predominantly participated in cellular, biological regulation, multicellular organismal, localization, signaling, multi-organism, and immune system processes. Protein-protein interaction (PPI) implied diverse clusters of the DEPs. Besides, the paralleled changes of the common upregulated and downregulated DEPs existed in both the liver and serum were validated in the mRNA expression of NRP1, MUP3, SERPINA1E, ALPL, and ALDOB as observed in our proteomic screening. Methods: We conducted hepatic and serum proteomic analysis based on the leptin-receptor-deficient mouse (db/db), a well-established diabetic mouse model with overt obesity and NAFLD. The results show differentially expressed proteins (DEPs) in hepatic and serum proteomic analysis. A parallel reaction monitor (PRM) confirmed the authenticity of the selected DEPs. Conclusion: These results are supposed to offer sensitive non-invasive serum biomarkers for diabetes and NAFLD.