Background and Aims: DNA constructs containing HCV antigens have become one of the vaccine candidates for induction of anti-HCV cellular and humoral immunity. In this study, we constructed a novel expressing vector harboring a fusion sequence derived from an overlapping fragment in the middle of NS3 and a truncated core fragment to avoid troubles reported to be associated with full gene expression. Methods: The partial NS3 (pNS3) and core genes were amplified by RT-PCR method using serum of HCV infected patient harboring genotype 1a of virus. After purification and cloning the genes into TA-cloning vector, they were evaluated by sequencing and restriction digestion analysis. The resultant pNS3 and core gene subcloned into expression vector separately followed by expression evaluation using RT-PCR and western blotting. The core expressing vector exploited for amplification of a new truncated core (50-160aa) sequence using PCR. Truncated core fragment was first cloned into TA vector at a natural restriction site downstream of pNS3 fragment. The resulting fused sequence was cut and subcloned into expression vector. The integrity and ability of expression of this fused sequence was evaluated by sequencing followed by RT-PCR analysis after DNA transfection into 293 cells. Results: The repeated sequencing data showed sequence integrity among the gene fragments as well as homology among them and reference 1a sequences. The colony-PCR, RT-PCR and western blotting confirmed insertion of genes into expressing vector, expression of genes in 293 cell line and production of protein in 293 respectively. Conclusion: This new expressing vector harboring a novel fused fragments of NS3 and core genes may overcome shortcomings in vaccine design in the setting of HCV disease.