Objective:We sought to produce a human specific anti-rabies-virus bivalent antibody with neutralizing activity by using the dimer effect of RII’s dimerization and docking domain(DDD)in protein kinase A(PKA). Methods:Linker-C-DDD sequence was optimized and synthesized. Fd section and light chain variable region (Vκ) of anti-rabies virus Fab094 were amplified. Fd section of Fab094 and linker-C-DDD gene were recombined using overlap PCR. Fd-DDD and Vκ were cloned into eukaryotic expression vectors and both vectors were transfected into 293 free style cells. Fab094-DDD antibody was expressed and purified. The immunological features of Fab094-DDD was detected by SDS-PAGE,Western blot assay,enzyme-linked immunosorbent assay(ELISA),co-IP,affinity test and indirect immunofluorescence test. The neutralizing activity of Fab094-DDD was detected by fluorescent antibody virus neutralization test. Results:The results demonstrated that the eukaryotic expression vectors of the human specific anti-rabies virus bivalent Fab094-DDD antibody were successfully constructed. The antibody effectively recognized the antigen and specifically combined the rabies virus,and the neutralization titer of the antibody was 213.2 U/mg. Conclusion:We successfully generated a bivalent Fab094-DDD antibody against rabies virus,and the antibody showed high neutralizing activity. Fab094-DDD antibody could establish the foundation for the treatment of rabies and could be applied to the construction of other bispecific human specific antibodies.