ABSTRACT Streptococcus agalactiae (Group B Streptococcus, GBS) is a multi-host pathogen, even causing life-threatening infections in newborns. Vaccination with GBS crossed serotypes vaccine is one of the best options for long-term infection control. Here we built a comprehensive in silico epitope-prediction workflow pipeline to design a multivalent multiepitope-based subunit vaccine containing 11 epitopes against Streptococcus agalactiae (MVSA). All epitopes in MVSA came from the proteins which were antigenic-confirmed, virulent-associated, surface-exposed and conserved in ten GBS serotypes. The in-silico analysis showed MVSA had potential to evoke strong immune responses and enable worldwide population coverage. To validate MVSA protection efficacy against GBS infection, immune protection experiments were performed in a mouse model. Importantly, MVSA induced a high titre of antibodies, significant proliferation of mice splenocytes and elicited strong protection against lethal-dose challenge with a survival rate of 100% in mice after three vaccinations. Meanwhile, the polyclonal antibody against MVSA did not only inhibit for growth of GBS from six crucial serotypes in vitro, but also protect 100% naive mice from GBS lethal challenge. These active and passive immunity assay results suggested that MVSA could therefore be an efficacious multi-epitope vaccine against GBS infection.