Nanovaccines based on self?assembling nanoparticles (NPs) can show conformational epitopes of antigens and they have high immunogenicity. In addition, flagellin, as a biological immune enhancer, can be fused with an antigen to considerably enhance the immune effect of antigens. In improving the immunogenicity and stability of a foot?and?mouth disease virus (FMDV) antigen, novel FMDV NP antigens were prepared by covalently coupling the VP1 protein and truncated flagellin containing only N?terminus D0 and D1 (N?terminal aa 1–99, nFLiC) with self?assembling NPs (i301).The results showed that the fusion proteins VP1?i301 and VP1?i301?nFLiC can assemble into NPs with high thermal tolerance and stability, obtain high cell uptake efficiency, and upregulate marker molecules and immune?stimulating cytokines in vitro. In addition, compared with monomeric VP1 antigen, high?level cytokines were stimulated with VP1?i301 and VP1?i301?nFLiC nanovaccines in guinea pigs, to provide clinical protection against viral infection comparable to an inactivated vaccine. This study provides new insight for the development of a novel FMD vaccine.