The first fluorescent probe for mammalian thioredoxin reductase (TrxR), TRFS-green, was designed, synthesized, and fully evaluated. The probe features a 1,2-dithiolane scaffold with a quenched naphthalimide fluorophore. TRFS-green displays a green fluorescence off on change induced by the TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore. It was demonstrated in vitro that TRFS-green manifests high selectivity toward TrxR over other related enzymes and various small molecule thiols as well as biological reducing molecules. HPLC analyses indicated that TRFS-green was exclusively converted to naphthalimide catalyzed by TrxR. The ability in triggering on the fluorescence signal by cellular protein extracts correlates well with the endogenous TrxR activity in different cells. Furthermore, inhibition of TrxR by 2,4-dinitrochlorobenzene or depletion of TrxR by immunoprecipitation remarkably decreases the reduction of TRFS-green by cellular protein extracts. Finally, TRFS-green was successfully applied in imaging TrxR activity in living cells. The fluorescence signal of TRFS-green in living cells was inhibited by pretreating the cells with TrxR inhibitor in a dose-dependent manner, potentiating the development of living cell-based screening assay for identifying TrxR inhibitors. We expect the novel fluorescent probe TRFS-green would facilitate the discovery of TrxR-targeting small molecules for potential therapeutic agents and provide significant advances in understanding the physiological/pathophysiological functions of TrxR in vivo.