Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae that has caused severe economic losses in many countries of the world. Regular vaccinations have been effectively used to control foot-and-mouth disease (FMD) in countries where the disease is enzootic. Distinguishing between infected and vaccinated animals in herds after immunization is an important component of effective eradication strategies. Nonstructural protein (NSP) 3B of FMDV is part of a larger antigen that is used for this differential diagnosis. In this study, an FMDV serotype-independent monoclonal antibody (MAb) against NSP 3B, 5D12, was generated. Using western blot, it was revealed that MAb 5D12 binds to three fragments of 3B displaying the motifs G1PYAGPLERQKPLK14, K18LPQQEGPYAGPMER32 and V45KEGPYEGPVKKPVA59. The motif G1PYAGPLERQKPLK14 was chosen for further mapping. Different truncated motifs derived from the motif G1PYAGPLERQKPLK14 were expressed as GST-fusion constructs for western blot analysis. The results showed that the 5-aa peptide P2YAGP6 was the minimal epitope reactive to MAb 5D12. Subsequent alanine-scanning mutagenesis analysis revealed that Pro2, Gly5 and Pro6 were crucial for MAb 5D12 binding to P2YAGP6. Furthermore, through sequence alignment analysis, the epitope PxxGP recognized by 5D12 was found to be present not only in 3B-1 but also in 3B2 and 3B3 and was highly conserved in seven serotypes of FMDV strains. Western blot analysis also revealed that the peptide epitope could be recognized by sera from FMDV-infected pigs and cattle. Thus, the 5D12-recognized 3B epitope identified here provides theoretical support for the development of MAb 5D12 as a differential diagnosis reagent for FMDV infection.