The incidence of hepatocellular carcinoma (HCC) is highest among primary liver cancer. HBV and HBV-induced liver cirrhosis may lead to HCC (1). At present, it is difficult to diagnose HCC at early stage or to differentiate HCC. Therefore, it is much needed to explore and develop a simple, rapid diagnostic method, which has higher sensitivity and specificity for HCC at early stage (2, 3). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel non-electrophoresis-based proteomic technology. SELDI offers the advantages of rapid and simple examination as well as high specificity and sensitivity (4, 5). To our knowledge,there has been little study reported using SELDI-TOF-MS technology to investigate HCC. In this study, 25 cases of HCC patients not receiving any therapy, 25 cases receiving the interposition chemotherapy and 50 cases of the healthy people were tested by Weak cationic exchange (WCX2) protein chip and SELDI-TOF-MS. The differentially expressed peptides or proteins were analyzed by BioMarker Wizard software. At the different M/Z value range, seven peptides/ proteins were obviously different among these three groups. Four peptides including 6489.48Da, 6662.34Da, 8593.75Da and 8720.23Da were up-regulated in healthy controls, two including 7777.27Da and 9250.00Da were up-regulated in the patients with HCC without receiving any therapy and one protein of 16200.17Da was up-regulated in the patients with HCC receiving the interposition chemotherapy. Using Biomarker Pattern software, one pattern including two peptides (7777.27Da, 9250.00Da) can separate HCC without receiving any therapy from normal controls. This diagnosis pattern gave the much-improved sensitivity of 92% and the specificity of 100%. Through searching protein database, these seven peptides or proteins were identified as possible Galanin related peptide, Pro-neuregulin-4 protein, small inducible cytokine A15 precursor, 9 kDa protein, CSL-zincfinger protein 1, mitochondrial hinge protein, actin related protein, respectively. Using SELDI-TOF MS, the method of sieving the tumor markers from HCC becomes quick and valid. These differentially expressed peptides or proteins could be biomarkers of HCC in the serum and drug targets for treating HCC.