The first near-infrared fluorescent probe with excellent water-solubility for gamma-glutamyl transpeptidase (GGT) has been developed by combining glutathione (GSH) as a recognition unit with a near-infrared hemicyanine fluorophore through an acrylyl linker. The probe exhibits a highly selective and sensitive fluorescent off-on response to GGT with a detection limit of 0.50 U/L, and the response mechanism is based on the enzyme-catalyzed cleavage of the gamma-glutamyl bond of GSH, followed by the spontaneous intramolecular cyclization and the release of the fluorophore. Notably, the probe has been used to image GGT in zebrafish and evaluate the inhibition ability of three common inhibitors of GGT both in vitro and in vivo, revealing that their inhibition efficiencies are acivicin > 6-diazo-5-oxo-L-norleucine > L-serineborate complex, and their corresponding IC50 values are 0.11 +/- 0.01 mM, 0.34 +/- 0.04 mM and 2.06 +/- 0.24 mM, respectively. The proposed probe is simple, and may have great potential for screening GGT inhibitors. (C) 2016 Elsevier B.V. All rights reserved.