Ultraperformance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC/Q-TOF MS) is an effective and sensitive analytical tool. A UPLC/Q-TOF MS-based metabonomics technique was employed to investigate sera from 40 patients with intestinal fistula and 17 healthy volunteers in an effort to find potential biomarkers of the disease and reveal their pathophysiological changes. After the UPLC/Q-TOF analysis, the retention time and m/z data pair for each peak were detected. Partial least squares discriminant analysis (PLS-DA) and coefficient of correlation analysis were used for marker selection and identification. According to the data, nine potential biomarkers were identified: glycochenodeoxycholic acid, glycocholic [corrected] acid, taurochenodexycholic acid, taurodeoxycholic acid, and two kinds of lysophosphatidyl choline (C16:0 and C18:2) were found with increased concentrations in the patients, and phenylalanine, tryptophan, and carnitine were found with decreased concentrations in the patients. The results suggested that a subclinical hepatic injury and abnormal metabolism of two essential amino acids (phenylalanine and tryptophan), and a key compound of fatty acid synthesis and beta-oxidation (carnitine), occurred in the fistula patients. This work demonstrates the utility of metabonomics as a top-down systems biology tool for understanding clinical problems.