Detection of glutathione in human serum is of great importance for clinical diagnosis of various diseases, such as AIDS, diabetes mellitus, Alzheimer disease and cancer. In this work, a new water-soluble bismacrocyclic polyamine-derived compound, namely L, which contains two molecules of 4-nitro-1,2,3-benzoxa-diazole as the fluorophores, was designed and prepared. The experiments of selectivity of L toward metal ions showed it could rapidly and sensitively detect Hg2+ with a detection limit of 27 nM. Furthermore, the cell imaging and co-staining experiments in HeLa cells demonstrated that the L-Hg2+ probe had selectivity for the Golgi apparatus to a certain degree. Moreover, it had excellent selectivity for biothiols, especially for glutathione. Finally, the probe was successfully applied to sensitively detect glutathione (GSH) in human serum and fetal bovine serum.