The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.