Over　the　past　few　decades,　pretargeted　bioimaging　has　made　significant　contributions　to　disease　diagnosis　and　the　real-time　visualization　of　biological　processes.　Notably,　the　inverse　electron　demand　Diels-Alder　(IEDDA)　reaction　between　tetrazine　(Tz)　and　trans-cyclooctene　(TCO)　has　shown　enormous　potential　in　pretargeted　bioimaging　applications　by　capitalizing　on　its　rapid　kinetics,　specific　reactivity,　biorthogonality　and　biocompatibility.　To　date,　imaging　probes　based　on　the　Tz−TCO　click　reaction　have　been　extensively　developed　and　widely　used　for　pretargeted　bioimaging　applications.　In　this　review,　we　focus　on　the　Tz−TCO　click　reaction　and　summarize　its　applications　in　pretargeted　bioimaging.　Firstly,　the　fundamental　principles　for　designing　imaging　probes　based　on　the　Tz−TCO　click　reaction　are　explained.　Then,　recent　advances　in　imaging　probes　based　on　the　Tz−TCO　click　reaction　are　discussed　in　detail.　Finally,　the　current　challenges　and　perspectives　of　the　Tz−TCO　click　reaction　in　pretargeted　bioimaging　applications　are　presented.　©　2023　Wiley-VCH　GmbH.