Hypochlorous acid (HOCl), as an important reactive oxygen species (ROS), plays key roles in normal physiological activities. Simultaneously, HOCl is also an efficient and strong oxidant, and is widely used in disease prevention and disinfection. Nevertheless, abnormal expression or excessive intake of HOCl may cause some serious diseases. Accordingly, the high sensitivity and specificity detection of HOCl in organisms and the environment is significant for understanding its roles in biology and environmental protection. Herein, a novel responsive 1,8-naphthalimide-based fluorescence probe PNL for the detection of HOCl was reported by a novel recognition mechanism. In the presence of HOCl, the PET effect of PNL was inhibited by the HOCl-induced cyclization reaction, leading to the “turn-on” fluorescence response. The recognition mechanism of PNL towards HOCl was determined by HR-MS, and the fluorescence “turn-on” sensing mechanism was rationalized via DFT calculation. PNL possesses the advantages of short response time (10 s), excellent selectivity, low cytotoxicity and reliability at a wide range of pH (3.0–11.5). Furthermore, the quantitative determinations of HOCl in practical water samples were achieved via the smartphone using PNL as the probe. Most importantly, the applications of PNL in fluorescent imaging of HOCl in vitro and the visualization of HOCl in an inflammatory model were successfully demonstrated. The development of PNL would also provide more abundant design strategies for the construction of HOCl fluorescence probes in the future.