Hypochlorous acid (HOCl) is crucial in various physiological processes, and monitoring its level in organisms is essential for understanding its biological functions and pathological effects. In this study, we developed a novel fluorescent probe based on dicyclohexylphosphine for rapid and sensitive detection of HOCl in an aqueous solution. The probe exhibits a significant fluorescence turn-on response to HOCl through the specific oxidative reaction between dicyclohexylphosphine and HOCl. It possesses high selectivity, instantaneous response (approximately 1 s), and a low detection limit of 15.3 nM. Moreover, biological imaging results demonstrated that the probe could be used for real-time fluorescence imaging of HOCl in living cells and zebrafish. The development of this probe may provide new insights into designing phosphine-based fluorescent probes for detecting oxidative species.