Abstract: Objective: Foot-and-mouth disease virus (FMDV) is an acute and highly contagious virus that infects cloven-hoofed domesticated ruminants. Discovery of effective T-helper (Th) epitopes is critical for the formation of stable interactions with Major Histocompatibility Complex (MHC) molecules and the induction of an adaptive immune response specific to the pathogen. Methodology: In this study, we sought to discover potential Th peptides that more broadly recognize supertypes of bovine leukocyte antigen (BoLA) using immuno-informatics approaches, including the IEDB database and ProPred tools. The peptide VP1Th was used to generate a combinatorial peptide library, VP1Thcomb, which mimicked population-spanning. Results: Th epitopes based on the substitution of amino acids at optimal anchors for peptide-MHC interactions that exhibited diverse binding pockets in the 130 polymorphic BoLA-DR3 alleles. Additionally, the primary and auxiliary anchor residues were improved to increase the ligand affinity of binding MHC molecules by substituting arginine at anchor residue 2 and to extend the peptide length at both ends of the Th epitope binding core region, respectively. Finally, the epitope-based vaccine VP1Thcomb-FMDV was chemically synthesized and vaccine potency reached 12.51 and 8.05 bovine PD50 per single dose administration challenged with the two serotypes of 10,000 BID50 FMDV, Asia 1 and O, respectively. Conclusion: Strong associations were also observed between virus neutralization test titers and protection. In conclusion, the strategy of epitope-based vaccine development provided a minimal structure of well-defined antigen to stimulate protective immunity against FMDV.