BACKGROUND: canine distemper (CD) is a highly contagious and fatal disease caused by canine distemper virus (CDV), posing a significant threat to carnivores. New CDV strain circulation and multi-species infection may lead to the potential dilemma of safety concern and insufficient efficacy of the commercial modified live vaccines. Safe and effective vaccines for canine and wildlife prevention of CD need to be continuously updated and developed. METHODS: we developed two DNA vaccines, p17F-LNP and p17H-LNP, encoding the fusion protein (F) or hemagglutinin protein (H) of a field CDV strain (HLJ17) and encapsulated in lipid nanoparticles (LNPs). Serum neutralizing antibody (NAb) was evaluated via neutralization tests, and mouse serum cytokine detection were evaluated via ELISA. RESULTS: immunization of p17F-LNP and p17H-LNP monovalent or bivalent were safe, and induced robust CDV NAb and cytokine responses in mice. LNP encapsulation improved immune responses compared to naked DNA formulation, and the bivalent formulation of p17F-LNP and p17H-LNP (p17F/H-LNP) exhibited synergistic effects with a high level of immune responses. Moreover, two doses of p17F/H-LNP induced long-lasting CDV NAb for over 300 days in dogs, and prime and boost NAb responses in foxes and raccoon dogs. CONCLUSIONS: the preliminary findings provided here warrant further development of the p17F/H-LNP vaccine for animal targets against CDV infection.