Due　to　their　highly　significant　biological　activities,　in　vivo　monitoring　biothiols,　including　glutathione　(GSH),　cysteine　(Cys),　and　homocysteine　(Hcy),　draws　great　attention.　Among　them,　GSH　is　the　representative　nonprotein　thiol　inside　the　cell　with　the　high　abundance,　protecting　cells　from　oxidative　stress　and　toxins.　In　this　work,　we　developed　a　fluorescent　probe　NI　for　sensitive　mapping　endogenous　biothiol　distribution　in　zebrafish.　We　used　1,8-naphthalimide　scaffold　as　easy　modifiable　fluorophore　and　2-thiophenecarbonxyl　as　recognition　moiety.　After　the　cleavage　of　ester　bond　by　GSH　addition,　NI　exhibited　strong　emission　with　large　Stokes　shift　(110　nm)　and　its　LOD　is　0.120　μM.　We　also　studied　the　difference　of　kinetic　response　of　GSH　over　Cys　and　Hcy.　Finally,　the　described　probe　was　successfully　applied　in　sensing　endogenous　biothiol　in　zebrafish,　showing　its　potential　utility　in　vivo　imaging.　©　2019