Detection　of　glutathione　(GSH)　in　the　body　is　essential　to　accurately　map　the　redox　state　of　cells　and　real-time　visualization　of　physiological　and　pathological　conditions　in　vivo.　However,　traditional　fluorescence　(FL)　imaging　in　the　near-infrared　I　region　(NIR-I,　650–900 nm)　is　difficult　to　quantitively　visualize　GSH　in　vivo　due　to　the　tissue　autofluorescence　background　and　disastrous　photon　scattering.　Herein,　a　NIR-IIb　(1500–1700 nm)　nanoprobe　consisting　of　4-nitrophenol-Cy7　(NPh)　conjugated　lanthanide-based　downconversion　nanoparticles　(DCNP@NPh-PEG)　is　developed　for　in　vivo　ratiometric　imaging　of　GSH.　In　the　presence　of　GSH,　NPh　shows　responsively　enhanced　FL　emission　at　808 nm,　thus　enhancing　FL　signal　at　1550 nm　of　DCNPs　excited　by　808 nm　(F1550,　808Ex)　through　non-radiative　energy　transfer　(NRET)　effect,　while　the　fluorescence　of　DCNP　at　1550 nm　excited　by　980 nm　laser　(F1550,　980Ex)　is　stable　because　no　NRET　occurred.　The　ratiometric　F1550,　980Ex/F1550,　808Ex　value　exhibits　a　linearship　with　GSH　concentration　ranged　from　0–24 mm　with　detection　limit　of　0.3 mm.　The　NIR-IIb　nanoprobe　has　excellent　performance　in　detecting　and　imaging　GSH　in　both　subcutaneous　tumor　and　orthotopic　colon　tumor　in　vivo　with　high　accuracy　and　resolution.　The　design　strategy　of　the　ratiometric　NIR-II　FL　nanoprobe　based　on　the　activated　FERT　effect　provides　a　reliable　tool　for　the　development　of　NIR-II　nanoprobes　for　accurate　biosensing　in　vivo.　©　2021　Wiley-VCH　GmbH