The invention relates to the fields of biotechnology and animal epidemic disease diagnosis and specifically discloses a kit for detecting an ORT as well as a preparation method and application thereof. Lipopolysaccharide of a Type B ORT serves as a coating antigen; the purified antigen has strong specificity and sensitivity for the ORT and can be used for detecting eighteen serotype ORTs at a time, and the antibody level of ORTs in chicken flocks can be truly and accurately reflected. In the invention, rabbit anti-avian influenza IgG marked by HRP serves as an enzyme-labeled antibody, and thekit can be widely used for detecting the universality for poultry such as chicken, ducks, geese and the like, so that the universality of the kit is improved.