Hand-foot-and-mouth disease (HFMD) is an infectious disease that usually occurs in children under five years of age. Mainly caused by Coxsackievirus A16 (CA16) or Enterovirus 71 (EV71). Hand-foot-and-mouth disease (HFMD) caused by the CA16 infection has mostly mild symptoms. However, if the cause of EV71 appears, serious neurological complications such as meningitis and encephalitis may occur. But there is no vaccine and medicines for EV71 infection yet. Therefore, to prevent EV71, multi-epitope vaccine research based on peptide amphiphiles (PAs) was conducted. For research purposes, epitope was selected in capsid protein of EV71, and the form of conjugated fatty acids of different lengths was synthesized. The nanofiber type immune stimulator was formed through self-assembly of each composed PAs in aqueous condition. To evaluate the immunogenicity and efficacy for a PAs-based immune stimulator, constructed PAs were injection route of subcutaneous (s.c.) into Balb/c female mice. One week after the last injection, serum and spleen samples were collected. Through ELISA and the Western blot, it was confirmed antigen specific to the particles of EV71. In addition, cytokine reactions formed by stimulating splenocytes were measured (IFN-γ, IL-2, IL-10, IL-12 and IL-17). Stimulation index was confirmed through MTT assay after treating antigen in splenocyte. In conclusion, this study reported that the immune response induced by multi-epitope vaccine based on peptide amphiphiles induces improved immunity from peptide and single peptide-amphiphiles. Thus, peptide amphiphiles-based multi-epitope vaccines might be a new approach that could improve the low immunogenicity of conventional peptide vaccines.