The present invention provides a method for purifying recombinant coxsackievirus A16 (CA16) virus-like particles, an application thereof in an vaccine, and the vaccine. The method comprises carrying out high density fermentation culture of recombinantly expressed engineering bacteria and methanol-induced expression of CA16 viroid granulin, carrying out centrifugation for collecting thalli, performing high-pressure homogenization and breaking, and purifying the supernatant by ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography and molecular sieve chromatography and the like. The CA16 viroid particle vaccine provided by the invention has good immunogenicity, safety, immunological characteristics and biological activity. The process is simple, and the purification is performed by chromatographic methods. The purification method is more conducive to linear amplification compared with density gradient centrifugation, and can be used in large-scale preparation and purification, and a high-purity (more than 99%) virus-like particle (VLP) stock solution can be obtained and can be used to prepare a vaccine for preventing CA16 infection, and has good economic value and application prospects.