Abstract: Objective: To clone and analyze the HCV-1b gene fragments isolated by Restriction Display technique（RD） Methods: Restriction enzyme Sau3A Ⅰwas chosen to digest the full-length HCV-1b cDNAs. The products were classified and re-amplified by RD-PCR, the RD-PCR fragments were purified and cloned into the pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. Results: The target HCV gene fragment ranging from 200 to 1000bp were isolated and sequenced which proved to be gene fragments derived from the HCV-1b genome. A total of 20 different cDNA fragments were quickly obtained. Conclusion: RD technique is of great value in obtaining a large number of size-comparable gene probes, which are suitable for the construction of cDNA fragments library and preparation of microarray probes.