VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-in-dependent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to (6)ETTLLE(11) at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif (6)ETTLLE(11) of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide (2)KKTEETTLLEDR(13) was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr(7), Thr(8), and Leu(10) are the functional residues of the 4B2 epitope Glu(6) and Leu(9) are required residues, and Glu(11) plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.