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.　The　Tz-TCO　click　reaction-based　pretargeting　bioimaging　strategy　involves　two　steps:　(1)　Enrichment　of　the　first　bioorthogonal　component　(Tz/TCO　moiety)　at　targets　of　interest　(TOI)　by　using　a　functional　group-modified　Tz/TCO　moiety;　(2)　addition　of　the　second　bioorthogonal　component　(TCO/Tz　motif)　for　the　in　situ　Tz-TCO　click　reaction,　which　is　accompanied　by　the　acquisition　of　imaging　signal　at　TOI.image