The invention discloses an EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and a preparation method and application thereof. A hand-foot-and-mouth disease virus is separated, whole genome sequencing is conducted to obtain EV71 virus VP1 structural protein, and the EV71 virus VP1 structural protein is divided into 20 groups to be synthesized into polypeptide segments. Serum containing an EV71 virus antibody is screened out, and No.2, No.4 and No.8 polypeptides with higher immune effects are initially screened out through an enzyme linked immune absorbent assay. In order to further verify whether the initially screened polypeptides have the immune effect in a mouse model, the invention reasonably designs a Balb/c mouse test model, the Balb/c mouse test model is subjected to pathological section HE dyeing pathological analysis, the No.8 polypeptide has the higher immune effect on the small intestine, the skeletal muscle and the brain of the model Balb/c mouse, and the No.2 polypeptide has the higher immune effect on the skeletal muscle of the model Balb/c mouse. Through the enzyme-linked immunosorbent assay and the pathological examination comprehensive analysis and verification of the model Balb/c mouse, a result indicates that the optimized No.2 polypeptide and No.8 polypeptide have huge potential in development and application of the EV71 virus vaccine.