Abstract: In order to develop a recombinant adenoviral vaccine, genes of a whole ORF coding genes of foot-and-mouth disease virus (FMDV) were amplified by RT-PCR and subsequently cloned into the vector pAdTrack-CMV to construct the recombinant adenoviral pAdTrack-CMV-ORF. PCR amplification and digestion with restriction endonucleases confirmed that the cloned ORF coding genes shared 99.8% similarity with that of original virulent strain type FMDV. Then the shuttle plasmid was linearized and transformed into competent cells of BJ5183 which containing the adenoviral backbone plasmid pAdeasy-2. The recombinant adenoviral plasmid (pAd-ORF) was identified by PCR amplification and digestion with PacⅠ. In this paper, we cloned the whole ORF coding genes of O type FMDV and successfully constructed the recombinant adenoviral shuttle vector.