Introduction: Hepatitis C virus (HCV) is one of the major medical problems. Human and chimpanzees are the only specific hosts which are naturally susceptible to HCV infection. Mice and other common laboratory animals are resistant to the virus, hence HCV prophylactic and therapeutic researches are very difficult and challenging. HCV non-structural protein 3 (NS3) is one of the most attractive targets for developing novel anti-HCV therapies as it is essential for the viral replication. This study was designed to produce stable SP2/0 tumor cell lines expressing NS3 of HCV for future basic and vaccine studies. Methods: A lentivector expressing NS3, named PCDH-NS3, was constructed by cloning of NS3 cDNA into downstream of CMV promoter of pCDH-CMV-MCS-EF1-Puro-GFP. The constructed plasmid was co-transfected with pMD2.G plasmid which encodes envelope VSV G protein and psPAX2 packaging plasmid into HEK-293T cells. The lentivector-containing supernatant was collected every 12 h for 72 h and NS3-Lentivector was concentrated by ultracentrifugation. Titers of the NS3 lentivector were estimated using flow cytometry. The SP2/0 cells were then infected by NS3 lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select NS3 positive cells. A single transfected clone was obtained using limiting dilution. The 1st and 6th passages of the cells cultured in vitro were harvested and NS3 mRNA was detected for by RT-PC. Results: The results showed that NS3 expressing lentivector plasmids and the two other helper plasmids could be transfected into HEK293T efficiently and packaged successfully as a pseudo-lentivector. Finally, the detection of NS3 mRNA in the 1st and 6th passages of SP2/NS3 cells was confirmed by establishment of a stable cell line. Conclusion: SP2/0 Cell line with stable expression of NS3 can be used as a suitable tumor model to facilitate research on HCV vaccine in vitro and in mice model and it could be served as a valuable tool for pharmaceutical HCV research to pave the way for further research on NS3 vaccine function.