To prepare and evaluate the clinical application of protein-chip for different HCV antibody detection, 905 serum samples were collected from three different hospitals. The samples were detected by ELISA method and protein-chip method respectively. Inconsistent samples were proved by imported HCV RIBA diagnostic kit. The results showed that (1)In all 905 serum samples, 294 positive samples and 611 negative samples were detected by ELISA method. Among the 294 positive samples, 292 positive samples and 2 negative samples were detected by chimeric antigen on the protein-chip and 288 positive samples, 2 negative samples, 4 indefinite samples were detected by the 4 segments of HCV antigens on the protein-chip. Among the 611 negative samples, 611 samples show negative result detected by chimeric antigen on the protein-chip and the 4 segments of HCV antigens on the protein-chip.The coordinate ratio of positive results are 99.3% and 98.9% respectively compared with ELISA method. The coordinate ratio of negative result are the same 100%. The 6 samples that were positive detemined by ELISA method and non-positive by protein-chip method were detected with RIBA reagent. All the 6 samples show non-positive result. (2)290 samples were detected with RIBA reagent. Among the 290 samples, 104 samples showed positive result, 66 samples showed positive result to single antigen segment, and 120 samples showed negative result. The 290 samples were also detected by protein-chip method. Among the 290 samples, 103 samples showed positive result, 61 samples showed positive result to single antigen segment, and 126 samples showed negative result. The result shows high coordinate ratio between the two methods (P＞0.05). It can be concluded that protein-chip reagent for detection of different HCV antibodies has higher sensitivity and specific than ELISA method and has the same accuracy as imported RIBA reagent. It will be a convenient and low costs reagent for diagnosis of HCV.