In order to produce polyvalent vaccines based on single rVSV vector, we investigated the immunogenicity, antibody neutralizing activity, and antigenic determinant domain of Zaire Ebola's fragment MFL (aa 393-556) that contains furin site and internal fusion loop. Both the recombinant protein and the recombinant plasmid of fragment MFL elicited high levels of antibody, similar to those of Zaire Ebola GP (ZGP). The MFL fragment of ZGP also elicited high levels of neutralizing antibody and induced moderate cellular immune response in mice, as revealed by the proliferation and cytokine secretion of splenocytes. Through the analysis of the induction of neutralizing antibody by pVAX1-based recombinant plasmids that expressed truncated fragments of MFL, we found that the domain containing the internal fusion loop and the furin site was the major contributor of fragment MFL's immunogenicity. Furthermore, the rVSV-based bivalent vaccine expressing Sudan Ebola GP (SGP) and MFL fragment elicited efficient cross-immunity against ZGP and SGP with high levels of neutralizing antibody. Our results indicate that fragment MFL is an effective and novel antigen for the production of neutralizing antibody and polyvalent vaccines of Ebola virus.