Foot and Mouth Disease (FMD) is highly contagious disease among cloven-hoofed animals. FMD virus has structural and non-structural proteins. Vp1 is the most immunogenic structural peptides of FMD virus, applied for major vaccine studies. Objective: Construction of Pet28-VP1 cassette for FMD virus type O/IRN/2010 and expression VP1 peptide as the most immunogenic antigen was the aim of this study. Methods: The FMD virus type O/IRN/2010 was isolated from cattle in Qom, Iran and propagated on IBRS2 and BHK21 cell lines. The VP1 gene was amplified using the specific primer pair and RT-PCR technique. The purified PCR product was sub-cloned into the unique BamHI and Xho1 cloning sites to construct the PTZ57R/T -VP1 cassette. The DH5&alpha strain was transformed with this cassette. The digested VP1 gene was cloned in the digested Pet28 and confirmed using double digestion. Then the Pet28-VP1 construction was transformed in BL21 strain. Results: Expression of VP1 peptide was evaluated by IPTG induction and SDS-PAGE and confirmed by Guinea pig specific polyclonal antibody against FMD virus type O and conjugated rabbit anti Guinea pig antibody- HRP. Also neutralizing antisera titre was protective for vaccinated animals by recombinant VP1 protein. Conclusion: Since the isolation of new FMD virus strains in different geographical locations and expression of VP1 peptide can be used in emergency and control settings as a recombinant vaccine in the same area. Therefore the Pet28-VP1 cassette was constructed in this study is a good candidate for preparation of peptide vaccine against FMD virus type O/IRN/2010 in the next future.