The present high mortality of lung cancer in China stems mainly from the lack of feasible, non-invasive and early disease detection biomarkers. Serum metabolomics profiling to reveal metabolic alterations could expedite the disease detection process and suggest those patients who are harboring disease. Using a nested case-control design, we applied ultra-high-performance liquid chromatography/mass spectrometry (LC-MS)-based serum metabolomics to reveal the metabolomic alterations and to indicate the presence of non-small cell lung cancer (NSCLC) using serum samples collected prior to disease diagnoses. The studied serum samples were collected from 41 patients before a NSCLC diagnosis (within 3.0 y) and 38 matched the cancer-free controls from the prospective Shanghai Suburban Adult Cohort. The NSCLC patients markedly presented cellular metabolism alterations in serum samples collected prior to their disease diagnoses compared with the cancer-free controls. In total, we identified 18 significantly expressed metabolites whose relative abundance showed either an upward or a downward trend, with most of them being lipid and lipid-like molecules, organic acids, and nitrogen compounds. Choline metabolism in cancer, sphingolipid, and glycerophospholipid metabolism emerged as the significant metabolic disturbance of NSCLC. The metabolites involved in these biological processes may be the distinctive features associated with NSCLC prior to a diagnosis.