Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes (152)FQEAFWLEDG(161), (168)LIYPHQ(173), and (46)DATSVD(51) reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope (152)FQEAFWLEDG(161) is exposed on surface of the virion and epitopes (46)DATSVD(51) and (168)LIYPHQ(173) are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope (168)LIYPHQ(173) is completely conserved in all BEV strains. Epitope (46)DATSVD(51) is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope (152)FQEAFWLEDG(161) is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.