The invention relates to an ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as a preparation method and application of the ELISA kit, and belongs to the field of biotechnology. The ELISA kit comprises an ELISA plate coated with a Cimaterol medicine antigen, a Cimaterol medicine monoclonal antibody, an enzyme marker, a Cimaterol standard sample solution, a substrate color developing solution, a stop solution, a concentrated cleaning solution and a sample diluent. The application of the ELISA kit mainly comprises the steps of sample pretreatment, operation and result analysis. The ELISA kit can be used for detecting Cimaterol medicine residue in animal tissues (muscles and livers), urine and fodders; no special instrument is required during pretreatment; the operation is simple and fast; the precision, accuracy, sensitivity, specificity and the like are high; the ELISA kit is suitable for screening and on-site monitoring of a great amount of samples.