Chemiluminescence　(CL)　with　the　elimination　of　excitation　light　and　minimal　autofluorescence　interference　has　been　wieldy　applied　in　biosensing　and　bioimaging.　However,　the　traditional　emission　of　CL　probes　was　mainly　in　the　range　of　400　to　650　nm,　leading　to　undesired　resolution　and　penetration　in　a　biological　object.　Therefore,　it　was　urgent　to　develop　CL　molecules　in　the　near-infrared　window　[NIR,　including　NIR-I　(650　to　900　nm)　and　near-infrared-II　(900　to　1,700　nm)],　coupled　with　unique　advantages　of　long-time　imaging,　sensitive　response,　and　high　resolution　at　depths　of　millimeters.　However,　no　NIR-II　CL　unimolecular　probe　has　been　reported　until　now.　Herein,　we　developed　an　H2S-activated　NIR-II　CL　probe　[chemiluminiscence　donor　950,　(CD-950)]　by　covalently　connecting　two　Schaap’s　dioxetane　donors　with　high　chemical　energy　to　a　NIR-II　fluorophore　acceptor　candidate　via　intramolecular　CL　resonance　energy　transfer　strategy,　thereby　achieving　high　efficiency　of　95%.　CD-950　exhibited　superior　capacity　including　long-duration　imaging　(~60　min),　deeper　tissue　penetration　(~10　mm),　and　specific　H2S　response　under　physiological　conditions.　More　importantly,　CD-950　showed　detection　capability　for　metformin-induced　hepatotoxicity　with　2.5-fold　higher　signal-to-background　ratios　than　that　of　NIR-II　fluorescence　mode.　The　unimolecular　NIR-II　CL　probe　holds　great　potential　for　the　evaluation　of　drug-induced　side　effects　by　tracking　its　metabolites　in　vivo,　further　facilitating　the　rational　design　of　novel　NIR-II　CL-based　detection　platforms.　Copyright　©　2023　the　Author(s).