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.