Objective To use metabolomics method to study the metabolic profiles of amino acids and lysophosphatidylcholine (LPC) in the serum of rats with nonalcoholic fatty liver disease (NAFLD), to identify biomarkers for NAFLD, and to speculate on the possible mechanism responsible for its occurrence. Methods NAFLD rats were prepared by feeding a high-fat diet and intraperitoneal injection of carbon tetrachloride. Levels of 15 LPCs and 18 amino acids in the serum were determined in control and NAFLD rats by liquid chromatography-mass spectrometry. Changes in serum LPC and amino acid metabolic profiles in NAFLD rats were analyzed by principal component analysis and orthogonal partial least squares discriminant analysis. Correlations between biomarkers and NAFLD were analyzed by Pearson’ s correlation analysis. Results The metabolic profiles of serum LPC and amino acids differed significantly between the NAFLD group and the control group and were completely distinct. LPC ( 20 ∶ 1), arginine, and glutamic acid had significant contributions to NAFLD and were identified as biomarkers. Furthermore, LPC ( 20 ∶ 1) and arginine were significantly correlated with serum biochemical indicators such as aspartate transaminase, alanine transaminase, low-density lipoprotein, and total bilirubin. Conclusions The metabolic profiles of serum LPC and amino acids may be closely related to NALFD.