Aim: Based on metabonomics, the metabolic markers of lung cancer patients were analyzed, combined with bioinformatics to explore the underlying disease mechanism. Materials & methods: Based on case–control design, using UPLC-Q-TOF/MS, urine metabolites were detected in discovery and validation set. Multivariate statistical analysis were performed to identify potential markers for lung cancer. A network analysis was constructed to integrate lung cancer disease targets with the above metabolic markers, and its possible mechanism and biological significance were explained. Results: A total of 35 potential markers were identified, 11 of which overlapped. Five key markers have a good linear correlation with serum biochemical indicators. Conclusion: The occurrence and development of lung cancer are closely related to disturbance of D-Glutamine and D-glutamate metabolism, amino acid imbalance. This test was registered on China clinical trial registration center ( www.chictr.org.cn/index.aspx ), registration number was ChiCTR1900025543.