Autoantibodies against tumor antigens represent one type of biomarker that may be assayed in serum for detection of cancer and monitoring of disease progression. In the present study, we used a proteomics-based approach to identify novel tumor antigens in non-small cell lung cancer (NSCLC). By combining two-dimensional electrophoresis, western blotting, mass spectrometry and enzyme-linked immunosorbent assay technology, we detected autoantibodies against α-enolase in a subset of NSCLC patientsʼ sera. When ‘Mean ODhealthy control sera + 3 SDhealthy control sera’ was used as the cut-off point, the prevalence of this autoantibody was 27.7% in patients with NSCLC (26 of 94), 1.7% in healthy control subjects (1 of 60), and not detectable in sera from 15 patients with small cell lung cancer, 18 patients with gastrointestinal cancer and nine patients with Mycobacterium avium complex infection of lung. Immunohistochemical staining showed that expression of α-enolase was increased in cancer tissues of NSCLC patients, and flow cytometric analysis confirmed the expression of α-enolase at the surface of cancer cells. The combined detection of autoantibodies against α-enolase, carcinoembryonic antigen and cytokeratin 19 fragment (CYFRA21-1) enhanced sensitivity for the diagnosis of NSCLC. Therefore, autoantibodies against α-enolase may constitute a promising biomarker for NSCLC. (Cancer Sci 2007; 98: 1234–1240)