Antisense RNA is an important field of antisense technique. In order to explore a novel approach for the treatment of hepatitis C virus (HCV) infection and prove the availability of transgenic cellular model HepG2. 9706, an antisense RNA was designed targeting at the highly conserved 5'NCR and translation initiation site of HCV RNA at nt positions 13-397. It was inserted into the downstream of SV40 promotor sequence of pGL3 control vector in which luciferase gene is deleted. The antisense RNA expression vector (pHCV-asR) was constructed and identified by PCR, endonucleases reaction and DNA sequencing. Its expression in transfected HepG2 cells was tested by RT-PCR. To evaluate the inhibitory activities of pHCV-asR, HepG2. 9706 cells were transfected using this construct via Lipofectin method. Luciferase activity in cell lysates was measured for quantitative determining antiviral effects within the cells. The results showed that the inserted sequence of the pHCV-asR is the same as the designed sequence and can express in HepG2 cells. It was also found that pHCV-asR in HepG2. 9706 has a dose-dependent inhibitory effects on luciferase expression controlled by HCV 5'NCR with a inhibitory rate of 57%.