A new fluorescent probe, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide (probe 3), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among biological thiols in aqueous media. Probe 3 is a nonfluorescent compound. On being mixed with biothiols under neutral aqueous conditions, the 2,4-dinitrobenzenesulfoyl moiety can be cleaved off by glutathione, and the blue emission of the pyrazoline at 464 nm is switched on, with a fluorescence enhancement of 488-fold for glutathione. Furthermore, probe 3 was highly selective for glutathione without interference from some biologically relevant analytes. The detection limit of glutathione was 4.11 × 10⁻⁷ M. The emission of the probe is pH independent in the physiological pH range. Moreover, the probe can be used for fluorescent imaging of cellular glutathione and can be used for detecting glutathione in calf serum.