The central carbon energy metabolism, which is crucial to the metabolic pathway in almost all living organisms and in the regulation of responses to various kinds of stress. Most of serious metabolic disorders in basic life activities of life are attributed to energy metabolism disorders. Metabolic dysfunction-associated fatty liver disease (MAFLD) and other metabolic disease are usually accompanied with the disturbance of energy metabolism. Maintaining energy homeostasis is crucial for overall health and even survival. To date, there is no systematic study on energy metabolites profile of MAFLD. In this study, we established a method for the simultaneous quantification of 31 endogenous metabolites involved three pathways in the tricarboxylic acid (TCA) cycle, glycolysis and oxidative phosphorylation metabolic pathway by using the UPLC-MS/MS system. This present method offers significant advantages including simple and fast preparation of a wide range of mice and human biological samples and was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability. Target metabolomics analysis was performed in different tissues and organs of mice and the substantial metabolic disparity among mice and human was demonstrated, further verifying the applicability and reliability of our method. Multivariate statistical analysis approach was conducted on 31 variables in human and mice serum samples. Based on VIP> 1, P value< 0.05, seven differential metabolites were screened out for MAFLD in human and mice. ROC analysis indicated that the seven potential markers provided a good diagnostic efficiency for MAFLD. The method demonstrated a simple and promising strategy to rapidly determine the energy metabolism status in mice and human, which can provide valuable results for mechanistic studies. This work provides metabolomic information for the study of metabolic diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to energy metabolism in medical institutions.