Hydrogen peroxide (H2O2) plays a vital role in various physiological and pathological processes. Thus, fluorescent probes of H2O2 are powerful tools for the investigation of H2O2-related diseases. However, developing fluorescent probes that do not irreversibly consume H2O2 presents a significant challenge. In this work, we introduce carbonate ester as a nonconsumptive recognizing molecule to construct RES-6C as a novel fluorescent probe of H2O2. RES-6C exhibited a selective and sensitive turn-on fluorescence response to H2O2, enabling the detection of H2O2 in cells without disturbing the cellular redox status. RES-6C has been applied to study nonalcoholic fatty liver disease, revealing that peroxisomes and mitochondria contribute to H2O2 production to a similar extent during very-long-chain fatty acid metabolism for the first time. It has also enabled fluorescent imaging of H2O2 in the LPS-induced inflammation mouse model. Overall, RES-6C serves as a versatile tool to monitor H2O2 in tissues and in vivo, providing new insights into the design of probes for H2O2.