To identify Zinc-alpha-2-glycoprotein (ZAG) expression in HCC for serum biomarker, by analyzing the serum proteome of the patients suffering from primary hepatocellular carcinoma (HCC), liver cirrhosis and healthy donors. The serum proteome of the patients from HCC, liver cirrhosis and healthy donors were separated and identified by two-dimensional electrophoresis. The differentially expressed proteins were analyzed by peptide mass fingerprint based on MALDI-TOF-MS and SWISS-PROT or BLAST nr database searching. RT-PCR and Western blotting analysis were used to confirm expression of ZAG in HCC. Five differentially expressed proteins were identified. Albumin, Serotransferrin, CD5 antigen-like precursor ( IgM - associated peptide) were down-regulated in HCC, ZAG and Ig gamma-1 chain C region were up-regulated in HCC. ZAG, a lipid mobilizing factor, is a member of the major histocompatibility complex (MHC) class I family of protein. Five proteins which were found differentially expressed in HCC provided useful information for screening diagnostic tumor markers of human HCC. ZAG might be a novel candidate serum biomarker for HCC early diagnosis.