Acute lung injury (ALI) is a diffuse inflammatory pulmonary damage caused by excessive ROS that break the coordination of normal physiological structures and functions. Hypochlorous acid (HOCl), one kind of ROS, is a hopeful biological marker for inflammation-related diseases. Therefore, the excessive generation of HOCl might be a significant reason for oxidative injury in ALI. Herein, we developed a fluorescent probe, namely BCy-HOCl, for quantitatively monitoring and visualizing HOCl in living cells and in vivo. The probe BCy-HOCl displayed a significant fluorescence signal enhancement towards HOCl with excellent selectivity and sensitivity. The variation of HOCl in the ALI cell model and ALI mouse model was evaluated with BCy-HOCl to clarify the relationship between ALI and HOCl. Our results verified that the HOCl levels conspicuously increased with the severity of the ALI. Thus, HOCl is likely to play a crucial part in the process of ALI, which will probably provide a new strategy for its treatment.