In this study, we used baculovirus to express hemagglutinin (HA) and neuraminidase (NA) to prepare a novel genotype of H3N2 canine influenza virus particles (VLPs). The effectiveness of the H3N2 VLP vaccine was evaluated by detecting HI antibodies, the antiviral protection rate, antibody persistence and anatomical examination of the lungs.A challenge model has been established in a previous study for the study of canine influenza virus-like particle vaccines. A/Canine/Shanghai/0103/2019, with a challenge dose of 106 EID50, infects 10-week-old healthy beagle dogs through nasal instillation and can cause severe clinical symptoms. Using a single dose of VLP vaccine for beagle dogs, the vaccine was tested at titers of 26 intranasally and 26 intramuscularly. One week after a single immunization, the HI titer promptly reached 28 among the immunized groups. The duration of antibody can persist for four months. We differentiated between CD4+ and CD8+ T cells in the peripheral blood. Four weeks after the single immunization, all beagles except those in the noninfected and nonimmunized groups were intranasally challenged with live H3N2 virus (1?×?106 EID50). All immunized beagles shed no virus at d 1–4 post-challenge. After the challenge, the placebo control beagles shed the virus on d 1 post-challenge (105.85±0.071 EID50). An anatomical examination of the lungs revealed that visible lesions were rarely detected in the lungs of the nasal immunization group, and the lungs were as healthy as those of the noninfected and nonimmunized groups were. The lung surfaces presented visible bleeding spots in the intramuscular immunization group and placebo-control group. Their effectiveness will provide a scientific basis for the promotion and use of these products.