Hydrogen peroxide (H2O2), an important reactive oxygen species (ROS) with unique destructive oxidation properties, can be produced in lysosomes to fight off pathogens. Although many fluorescent probes have been developed for the detection and imaging of H2O2, the development of a ratiometric fluorescent probe for H2O2 detection and imaging in lysosomes and an inflammation model remains rather scarce. Therefore, it is important to develop an efficient tool for monitoring H2O2 in inflamed tissues to evaluate the physiological and pathological relationship between inflammation and lysosomal H2O2. In this work, a new naphthalimide-based lysosome-targeting fluorescent probe (NPT-H2O2) for ratiometric detection and imaging was developed in vitro and in vivo. The probe exhibited two well-resolved emission peaks separated by 125 nm, rapid response (<40 s), and high selectivity and sensitivity toward H2O2, as well as low cytotoxicity in vitro. Inspired by prominent features of these results, we further successfully applied NPT-H2O2 for H2O2 imaging with a dual-channel in living cells, demonstrating that our probe NPT-H2O2 was targeted in the lysosomes. Finally, NPT-H2O2 was used for H2O2 detection in inflamed tissues and achieved satisfactory results. We predict that our probe can be used as a powerful tool to reveal the relationship between physiology and pathology of inflammation and lysosomal H2O2.