The invention provides polypeptide used for preparing O type peptide vaccine of cattle foot-and-mouth disease. The polypeptide comprises an amino acid sequence presented by SEQ ID NO.1 and the peptide vaccine containing the polypeptide. The O type synthetic peptide vaccine of cattle foot-and-mouth disease provided by the invention has excellent immune efficacy, and cannot cause the problems of fever, swelling and the like existing in traditional vaccines, so that the vaccine can effectively respond to antigenic variation of current foot-and-mouth disease virus, prevents biosecurity problems, is easy to synthesize in a large scale, and has an excellent application prospect. The invention further provides preparation methods and pharmaceutical applications of the polypeptide and the peptide vaccine.