Detection of FMDV non-structural protein 3D antibodies has been used as a complementary method for sero-epidemiological studies as an indirect indicator of FMDV infection. In order to develop a sensitive cELISA to detect FMDV antibodies, immune dominant epitopes in FMDV-3D protein were identified by peptide array analysis. Monoclonal antibodies were then raised to a selected epitope and used in cELISA. Ninety two peptides corresponding to the complete amino acid sequence of FMDV-3D were synthesized. The sera from 15 FMDV infected cows were tested for binding to the peptides in an indirect ELISA. One major peptide (3D-4) was recognized by antisera in 12 of the 15 infected cows (80%). The sequence was formed by amino acid residues 16-30 of FMDV-3D. The mAbs produced from the mice immunized with native 3D showed neither reactivity to this epitope nor competition with sera from FMDV infected cattle. However, the mAbs produced from the mice immunized with native 3D and boosted with the peptide 3D-4 showed reactivity with native 3D, recombinant 3D as well as competition with sera of FMDV infected cattle and sheep in ELISA assays. Immune response to FMDV-3D was determined using a cELISA. All cattle and sheep tested were positive at 9 dpi and remained positive until the end of the experiment on days 28-31 (>50% inhibition). This demonstrated that mAbs directed to the peptide 3D-4 were effective competitors to the polyclonal antibodies against 3D in infected sera. The approach described here provides a useful tool for specific mAb production in the development of new diagnostic tests.