Serum was obtained from 25 male renal cell carcinoma (RCC) patients and 21 healthy males. O-glycans were released by a β-elimination reaction and purified by graphitized carbon cartridge solid phase extraction, then profiled by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. After noise removal and peak alignment, 1372 peaks were extracted from 200000 data points. Feature peaks were analyzed by calculation of differential sensitivity and specificity. The combination of two feature peaks was chosen as a biomarker and could clearly differentiate RCC and normal samples in our study group.