The invention discloses an rT3 chemiluminiscence immunodetection kit as well as a detection method and application thereof and belongs to the technical field of detection of in vitro diagnosis. The detection kit comprises the following components: (1) a marker system which comprises a reverse triiodothyronine antigen or an anti-reverse triiodothyronine antibody which is directly or indirectly connected with a marker tracer, and (2) a magnetic microsphere system which comprises a magnetic microsphere which is correspondingly directly or indirectly connected with the anti-reverse triiodothyronine antibody or the reverse triiodothyronine antigen. In the rT3 chemiluminiscence immunodetection, a high-sensitivity chemiluminiscence determination technology is combined with a high-specificity immunoreaction, so that the rT3 chemiluminiscence immunodetection kit has the advantages of high sensitivity, good specificity and wide detection range. The rT3 chemiluminiscence immunodetection kit also can be matched with a chemiluminiscence analyzer in a conventional technology, so that the full-automatic detection is realized, experiment errors caused by manual factors are avoided, and the analysis efficiency is improved.