The invention provides EV71virus-like particles as well as preparation method and application of the EV71virus-like particles. The method comprises the following steps of: connecting a P1 protein gene and 3CD protease gene of an EV71 virus with predicated mean vote (PMV) plasmid to build PMV-P1-3CD recombinant expression plasmid; turning into hansenula polymorpha AU-0501 expression bacterial strain to obtain an AU-PMV-P1-3CD recombinant expression bacterial strain; fermenting and culturing the recombinant expression bacteria strain and performing inducible expression on EV71 virus-like particle proteins by using methanol; centrifugally gathering thallus to perform homogenate crushing at high pressure; and purifying the supernate through ion-exchange column chromatography, hydrophobic chromatography and sieve chromatography to obtain the EV71virus-like particles. EV71virus-like particle vaccines provided by the invention has high immunogenicity, security, immunological characteristics and biological activity; and the EV71virus-like particles can be prepared in a large scale and purified and used for preparing vaccines for preventing EV71 infection, and has good economic value and application prospect.