Glutathione　(GSH),　the　most　common　and　abundant　antioxidant　in　the　body,　is　particularly　concentrated　in　cancer　cells　(2–10　mM).　This　concentration　is　approximately　1000　times　that　of　normal　cells,　making　GSH　a　specific　tumor　marker.　Overexpression　of　GSH　is　critical　for　mapping　the　redox　state　of　cancer　cells.　However,　there　are　few　probes　and　detection　methods　responsive　to　GSH　that　can　quantitatively　visualize　GSH　in　vivo　in　two-photon　excitation　fluorescence　(TPEF)　imaging　mode.　The　experimental　results　show　that　TPEF-GSH　could　not　only　target　GSH　in　tumors,　but　also　establish　the　quantitative　relationship　between　TPEF　signal　and　GSH　concentration.　We　explored　the　optimal　two-photon　excitation　wavelength　of　TPEF-GSH,　the　optimal　cell　incubation　duration　with　TPEF-GSH,　the　best　imaging　time　point　for　GSH　in　cells,　and　the　quantitative　relationship　between　the　TPEF　signal　and　the　changes　in　GSH　concentrations.　In　zebrafish　embryo　and　zebrafish　experiments,　the　ratiometric　value　of　TPEF-GSH　increased　with　the　decrease　of　GSH　concentration.　Microinjection　and　co-incubation　were　used　to　verify　whether　the　ratiometric　value　could　quantify　endogenous　GSH　in　tumor-bearing　zebrafish,　and　the　obtained　GSH　levels　were　4.66　mM　and　5.16　mM,　respectively.　The　ratio　TPEF　probe　could　accurately　visualize　and　quantify　GSH　in　vivo,　reflecting　the　redox　status　of　the　tumor.　The　design　of　the　ratiometric　molecular　probe　provides　a　reliable　strategy　for　the　development　of　TPEF　nanoprobe　in　vivo.　In　this　article,　a　new　GSH　sensitive　molecular　probe,　TPEF-GSH,　has　been　developed　with　good　specificity　and　sensitivity.　TPEF-GSH　was　successfully　used　to　image　cancer　cells　in　vitro　and　tumor-bearing　zebrafish　in　vivo,　and　to　further　detect　GSH　levels.　©　2022　Wiley-VCH　GmbH.