The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research. Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E. coli. The purified protease could cleave the recombinant protein NS5ab into two parts. The purified protease was used as target to screen binding peptides from phage displayed peptide library. After three rounds of affinity screening, 37 out of 44 randomly selected phages could bind specifically with the single-chain serine protease and their specificity were verified by competitive ELISA. The 13 sequenced clones represents 6 kinds of sequences of which the amino acids composition is in bias and there is a consensus sequence: [H/F/W]-H-W-X-X-W.