Metabolomics enables the analysis of metabolites within an organism, which offers the closest direct measurement of the physiological activity of the organism, and has advanced efforts to characterize metabolic states, identify biomarkers, and investigate metabolic pathways. A high degree of innovation in analytical techniques has promoted the application of metabolomics, especially in the study of clinical surgery. Metabolomics can be employed as a clinical testing method to maximize therapeutic outcomes, and has been applied in rapid diagnosis of diseases, timely postoperative monitoring, prognostic assessment, and personalized medicine. This review focuses on the use of mass spectrometry and nuclear magnetic resonance-based metabolomics in clinical surgery, including identifying metabolic changes before and after surgery, finding disease-associated biomarkers, and exploring the potential of personalized therapy. Challenges and opportunities of metabolomics in organ transplantation are also discussed, with a particular emphasis on metabolomics in donor organ evaluation and protection, prognostic outcome prediction, as well as postoperative adverse reaction monitoring. In the end, current limitations of metabolomics in clinical surgery and future research directions are presented.