Hypochlorite (ClO−) plays a vital role in innate immune defense; however, elevated levels can induce oxidative stress and contribute to various pathologies. Herein, we present a naphthalimide-based “off–on” fluorescent probe, NP-OX, designed for the highly sensitive and specific detection of ClO− in biological systems. NP-OX incorporates a hydroxamic acid group for targeted recognition and reacts with ClO−, demonstrating exceptional analytical performance characterized by an ultra-low limit of detection (15 nM), rapid response kinetics (<20 s), exquisite selectivity against diverse biological interferents, and broad pH adaptability (pH 4.0–10.0). Due to its low cytotoxicity and excellent membrane permeability, NP-OX was effectively utilized for fluorescence imaging of both exogenous and myeloperoxidase (MPO)-catalyzed endogenous ClO− in living SiHa cells. Consequently, NP-OX represents a robust molecular tool for monitoring ClO− fluctuations in complex biological systems, providing significant potential for exploring its roles in immunity and pathology.