Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "(92)EYIEKA(97)" "(23)EGPYAGPLE(31)" and "(EPHH212)-E-209", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "(92)EYIEKA(97)" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "(23)EGPYAGPLE(31)" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "(23)EGPYAGPLE(31)" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.