Monkeypox (mpox) is a zoonotic viral disease transmitted by the monkeypox virus (MPXV). Viral protein-based nanovaccines hold promise in preventing the infection of MPXV. MPXV-derived antigens (A29L, A35R, and M1R) were capable of eliciting protective immunity and suffered from low immunogenicity. Adjuvants and delivery systems were critical to improving the immunogenicity of antigens. In this study, a pathogen-mimicking nanovaccine was developed by conjugation of poly(amidoamine) (PAMAM) dendrimers with deoxycholic acid to form cationic nanoparticles as a delivery platform. The three antigens were individually conjugated with carboxylated β-glucan, a polysaccharide adjuvant, and subsequently self-assembled onto dendrimer nanoparticles via electrostatic interactions. The resulting nanovaccine induced robust antigen-specific antibody production, stimulated splenic levels of Th1- and Th2-type cytokines, and enhanced secretion of IFN-γ and IL-4 by CD4+ and CD8+ T cells. Additionally, it promoted the maturation of dendritic cells, activated T and B cells, and enhanced cytotoxic T cell activity. Notably, the vaccine stimulated the formation of B and T memory cells, providing long-term immune protection. Crucially, the vaccine conferred cross-protection against the lethal ectromelia virus (ECTV) challenge in mice while exhibiting no significant organ toxicity. These findings suggest that the vaccine is a promising candidate to deal with life-threatening mpox infections.