Assembly of nucleocapsids is an attractive target for novel anti-hepatitis B virus (HBV) agents, and intracellular single-chain variable fragment (scFv) antibodies against HBV core (HBc) protein are a class of potential alternatives for this purpose; however, their application is limited by the lack of a suitable means of delivery. Owing to the favorable performance of cytoplasmic transduction peptide (CTP) in cargo delivery in hepatocytes, we purified an anti-HBc scFv fused to CTP using a previous screened sequence by a prokaryotic expression system and evaluated its efficacy in the inhibition of HBV in vitro. Our results showed that cytoplasmic translocation of the previous anti-HBc scFv was achieved by CTP in HepG2.2.15 cells. Immunoprecipitation analysis indicated the fusion protein anti-HBc scFv-CTP interacted with its target antigen HBc, and negligible cytotoxicity was observed. Moreover, the anti-HBc scFv-CTP interfered with nucleocapsid assembly and markedly reduced both the supernatant HBV DNA level and the intracellular DNA replication intermediates, with a 5.1 µM of half maximal effect concentration and a dose-dependent effect. In conclusion, this novel anti-HBc scFv fused to CTP demonstrated inhibitory activity of HBV replication in vitro and warrants further in vivo study.