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The endoplasmic reticulum (ER) has a central role in the fine-tuning of environmental and internal stimuli. We herein report a ratiometric fluorescent probe, α-Naph, capable of determining basal H2O2 in the ER. The probe specifically responds to H2O2. The limit of detection of the probe is as low as 38 nM, making it a feasible sensor to image intracellular basal H2O2. In addition, utilizing its ratiometric property, we are able to measure the concentration of H2O2 in the ER quantitatively, eliminating the error caused by the probe concentration and environment. The intracellular concentration of H2O2 in the ER is calculated to be 0.692 µM under normal conditions and 1.26 µM under the stimulation of phorbol myristate acetate. [ABSTRACT FROM AUTHOR]