Abstract: HCV core region, being most conserved, in the whole genome can be used as a diagnostic marker in enzyme linked immuno-sorbant assay development. However, quasi species nature of HCV during its replication in host is the major hindrance in developing an effective therapeutic agent. Recombinant fusion protein for HCV immuno-assay from core region was prepared by PCR amplification and subsequent cloning in expression vector PET32a which was expressed by iso-propyl thio-B-galactosides (IPTG) in E. coli strain DE3 followed by purification by affinity chromatography. Fusion protein as an antigen reacted with human antisera containing antibodies. This HCV specific immuno-assay development based on recombinant HCV core protein will be the foundation in developing antiviral immuno-assays in Pakistan.