The　development　of　probes　for　early　monitoring　tumor　therapy　response　may　greatly　benefit　the　promotion　of　photodynamic　therapy　(PDT)　efficacy.　Singlet　oxygen　(1O2)　generation　is　a　typical　indicator　for　evaluating　PDT　efficacy　in　cancer.　However,　most　existing　probes　cannot　quantitatively　detect　1O2　in　vivo　due　to　the　high　reactivity　and　transient　state,　and　thus　have　a　poor　correlation　with　PDT　response.　Herein,　a　1O2-responsive　theranostic　platform　comprising　thiophene-based　small　molecule　(2SeFT-PEG)　and　photosensitizer　Chlorin　e6　(Ce6)　micelles　for　real-time　monitoring　PDT　efficacy　is　developed.　After　laser　irradiation,　the　Ce6-produced　1O2　could　simultaneously　kill　cancer　and　trigger　2SeFT-PEG　to　produce　increased　chemiluminescence　(CL)　and　decreased　fluorescence　(FL)　signals　variation　at　1050 nm　in　the　second　near-infrared　(NIR-II,　950–1700 nm)　window.　Significantly,　the　ratiometric　NIR-II　CL/FL　imaging　at　1050 nm　could　effectively　quantify　and　monitor　the　concentration　of　1O2　and　O2　consumption　or　recovery,　so　as　to　evaluate　the　therapeutic　efficacy　of　PDT　in　vivo.　Hence,　this　1O2　activated　NIR-II　CL/FL　probe　provides　an　efficient　ratiometric　optical　imaging　platform　for　real-time　evaluating　PDT　effect　and　precisely　guiding　the　PDT　process　in　vivo.　©　2022　Wiley-VCH　GmbH.