Although post-exposure prophylaxis (PEP) can prevent rabies, the price and shortage of rabies immunoglobulin (RIG) makes PEP unaffordable and unavailable. Monoclonal antibodies (MAb) is a substitute for RIG and could even be therapeutics because the presence of virus neutralizing antibodies (VNA) in the central nervous system (CNS) can clear RABV. However, antibody molecule is too big to penetrate the Blood-brain Barrier (BBB). Single-chain variable fragment (scFv) consists of a peptide linker and variable regions of heavy and light chain and thus is a small molecule and might penetrate the BBB. In this study, a scFv was constructed from a potent rabies MAb, SO57, and showed neutralizing titer of 41.5 IU/mL, which is far beyond the protective titer of 0.5 IU/mL recommended by WHO. The results show that SO57 scFv is a promising substitute for RIG. Furthermore, its likely to enter the CNS and could be a therapeutics against rabies.