Cu2+ and Biothiols play a critical role in diverse biological processes in organisms. Herein, we reported using a well‐known ESPT molecule 2‐(2’‐Pyridyl)benzimidazol(PBI) as a fluorescent probe to detect Cu2+ and GSH in aqueous solution. PBI presented a rapid fluorescence “ON‐OFF” response on Cu2+ over other metal cations, with a low detection limit (39.4 nM), and also been applied in real water samples detection. Through mass spectroscopy, single crystal X‐ray diffraction and DFT calculation, we confirmed that two PBI molecules would form a stable complex with one Cu2+. After adding GSH, PBI‐Cu2+ complex would release the free probe, and the “on‐off‐on” recognition process to achieve continuous detection of Cu2+ and GSH.