Chemiluminescence　(CL)　is　a　promising　tool　for　diagnostics　and　sensing　due　to　its　inherent　excitation　light-free.　However,　most　CL　probes　emit　exclusively　visible　light　with　limited　penetration　depth,　hindering　their　broad　application　for　in　vivo　deep　tissue　imaging.　This　work　reports　an　activated　unimolecular　CL　probe　(CL-P)　that　emits　light　in　the　near-infrared　(NIR)　region　allowing　a　superior　real-time　visualization　and　detection　of　NAD(P)H　quinone　oxidoreductase　1　(NQO1)　in　vivo.　The　NQO1-specific　trimethyl-locked　quinone　is　a　lock　for　masking　the　NQO1　trigger　site,　and　it　was　conjugated　on　the　NIR　Schaap＇s　dioxetane　chemiluminophore,　achieving　not　only　a　subtle　signal-to-noise　ratio　even　at　a　depth　of　15　mm　in　the　chicken　breast　tissues,　but　also　sensitively　detecting　the　positive　expression　of　NQO1　in　A549　cells.　CL-P　has　been　successfully　used　for　visualizing　the　aberrantly　expressed　NQO1　in　subcutaneous　xenograft　A549　tumors.　To　our　knowledge,　this　is　the　first　work　reporting　the　detection　of　NQO1　activity　in　the　NIR　region　by　unimolecular　CL　mode.　Overall,　the　NQO1-activated　visualization　platform　provides　a　guideline　to　transform　a　unimolecular　CL　sensor　into　a　licensed　imaging　agent　for　the　in　vivo　detection　of　enzyme-related　diseases.　©　2022　SIOC,　CAS,　Shanghai,　&　WILEY-VCH　GmbH.