The Minute Virus of Canines (MVC), classified under the genus Bocaparvovirus, causes severe respiratory and gastrointestinal symptoms in neonatal canines worldwide. The structural protein VP2 is essential for the attachment, infection, uncoating, and induction of the host immunological response to MVC. This study aimed to prepare a monoclonal antibody (mAb) against VP2 using the hybridoma technique. AlphaFold and CavityPlus bioinformatics analysis revealed that the N-terminal region of VP2 (amino acids 1-300) possesses structural characteristics that make it the most suitable target for effective antibody generation. The recombinant plasmid pET-32a(+)-VP2(N300) with fused Trx and His tags was successfully constructed. After immunizing mice, nine hybridoma cell lines were obtained, namely 1G5, 1G5-1, 1I24, 1I24-1, 2E6-1, 2 N9, 3C12-1, 4 M1, and 4 M1-1. The ascitic antibody titers of all cell lines were above 1:100,000. Western blot analysis of Walter Reed canine cells infected with MVC indicated the selection of three strains of monoclonal antibodies with strong specificity: 1G5, 3C12-1, and 4 M1. These three strains can be employed in immunofluorescence (IF) and immunoprecipitation (IP) tests for detecting VP2 protein. The monoclonal antibody mAb VP2 prepared in this study may serve as a valuable tool for detecting MVC and beneficial for investigating the mechanisms of MVC infection.