Objective To analyze the serum metabolomics of non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus (T2D), and atherosclerosis (AS). Methods NAFLD, T2D, and AS mouse models were established, and serum metabolomics were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). Data were analyzed using the R language MetaboAnalystR package. Differential metabolites were screened by multivariate statistics. Differential metabolic pathways were obtained by untargeted KEGG enrichment analysis. The result were analyzed for correlations between differential metabolites and blood lipids. Results NAFLD, T2D and AS shared 48 differential metabolites that were particularly enriched in linoleic acid metabolism, pentose phosphate metabolism, arachidonic acid metabolism, and histidine metabolism pathways (P<0. 05). The differential metabolites were significantly correlated to blood lipids. Compared with the control group, the main specific metabolic pathways of NAFLD, T2D and AS were Darginine and D-ornithine metabolism (P=0. 09), galactose metabolism, primary bile acid biosynthesis, starch, and sucrose metabolism (P<0. 05), and sphingolipid and pyrimidine metabolism (P<0. 05), respectively. The three diseases were compared pairwise, and there were differences in D-arginine and D-ornithine metabolism (P=0. 12), starch and sucrose metabolism (P<0. 01), D-glutamine and D-glutamate metabolism (P<0. 05), synthesis and degradation of ketone bodies (P=0. 06), alanine, aspartate and glutamate metabolism (P<0. 05), sphingolipid metabolism (P<0. 05), valine, leucine and isoleucine degradation (P<0. 05), and glycine, serine, and threonine metabolism (P=0. 12). Conclusions This study revealed the common and specific metabolic characteristics of NAFLD, T2D and AS, and provides for a research direction for comprehensive prevention and individualized treatment of these diseases.