Development of fluorescent/luminescent probes for rapid, selective and sensitive detection of reactive oxygen species (ROS) is of great significance for understanding the roles of ROS in pathophysiological processes. In the present research, a visible-light-excitable Eu3+ complex-based probe, Eu(L)3(DPBT), is designed and synthesized for the time-gated luminescence (TGL) determination of hypochlorous acid (HClO) in vitro and in vivo. The proposed probe exhibits a rapid, selective and sensitive TGL response to HClO, and excellent localization of mitochondria in living cells with low cytotoxicity. These features allow the probe to be used for the TGL sensing and imaging of HClO formation in mimic inflammatory cells at a subcellular level, as well as in endotoxin-induced liver injury and rheumatoid arthritis in live mice. In addition, by immobilizing the probe in the PEG hydrogel, the smart sensor films with rapid response to HClO were prepared, and successfully used for the real-time monitoring of HClO generation in mouse wounds, in order to distinguish the infected wounds from acute ones. Overall, this study provides a useful tool for the clinical monitoring and treatment of wound diseases.