Lung cancer is a leading cause of cancer death worldwide, and very few specific biomarkers can be used in its clinical diagnosis. Using surface‐enhanced laser desorption–ionization time‐of‐flight mass spectrometry (MS) to find novel serum biomarkers for lung cancer, we analyzed 227 serum samples, including 146 lung cancers, 41 benign lung diseases and 40 normal individuals. Three peaks, at 13.78, 13.90 and 14.07 k m/z, were significantly lower in lung cancer sera compared with sera from normal individuals (P < 0.001), whereas these peaks were higher than those in the sera of benign lung diseases (P < 0.001). The peaks were identified as native transthyretin (TTR) and its two variants by one‐dimensional polyacrylamide gel electrophoresis, ESI‐MS/MS, immunoprecipitation and western blot analysis. An enzyme‐linked immunosorbent assay indicated that TTR levels were consistent with surface‐enhanced laser desorption–ionization analysis in all groups tested. It gave 78.5% sensitivity and 77.5% specificity for lung cancer versus normal at the cut‐off point 115 µg/mL, and 66.7% sensitivity and 64.4% specificity for lung cancer versus benign lung diseases at the cut‐off point 88.5 µg/mL. Therefore, TTR may be useful as a biomarker to improve the diagnosis of lung cancer. (Cancer Sci 2007; 98: 1617–1624)