Background Smoking is the leading cause of death in the world. This study focused on the difference of the serum proteomic profiling between healthy smokers and nonsmokers in order to find smoking-specific serum biomarkers. Methods Pattern-based proteomic profiling of 100 serum samples (from 50 Chinese male smokers and 50 matched nonsmokers) was performed through magnetic bead fractionation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and resulting data were statistically analyzed by Ciphergen Protein Chip software 3.0.2. Results We found 72 serum peaks were significantly different between smokers and nonsmokers (P <0.05). Marker peaks of mass-to-charge ratio (m/z) 3159.13, 7561.03 and 9407.32 were smoking-specific. Conclusion The preliminary data suggested that smoking-specific serum biomarkers could be detected in humans. Chin Med J 2012;125(17):3027-3032