The invention discloses a Senecavirus ELISA antibody detection kit and a preparation method and application. The Senecavirus ELISA antibody detection kit comprises a Senecavirus VP1 protein-precoatedELISA plate, a blocking solution, a diluted sample solution, an enzyme conjugate, a concentrated washing solution, an enzyme substrate solution and a stopping solution. Senecavirus VP1 protein is usedas a coating antigen for the first time, a kit capable of detecting Senecavirus is established, and the antibody level of the Senecavirus in serum can be rapidly detected; the detection kit is high in sensitivity, good in specificity, good in repeatability and stable in result; the detection kit can be applied to monitoring of the antibody level of the Senecavirus so as to understand the status of the Senecavirus antibody in a whole pig herd; in addition, the detection kit uses an Escherichia coli expression system, and has the advantages of economy and cheapness.