Chemiluminescent　probes　based　on　1,2-dioxetane　scaffold　are　one　of　the　most　sensitive　imaging　modalities　for　detecting　disease-related　biomarkers　and　can　obtain　more　accurate　biological　information　in　cells　and　in　vivo.　Due　to　the　elimination　of　external　light　excitation,　the　background　autofluorescence　problem　in　fluorescence　technology　can　be　effectively　avoided,　providing　ultrahigh　sensitivity　and　signal-to-noise　ratio　for　various　applications.　In　this　review,　we　highlight　a　comprehensive　but　concise　overview　of　activatable　1,2-dioxetane-based　chemiluminescent　probes　by　reporting　significant　advances　in　accurate　detection　and　bioimaging.　The　design　principles　and　applications　for　reactive　species,　enzymes,　and　other　disease-related　biomarkers　are　systematically　discussed　and　summarized.　The　challenges　and　potential　prospects　of　chemiluminescent　probes　are　also　discussed　to　further　promote　the　development　of　new　chemiluminescence　methods　for　biological　analysis　and　diagnosis.