A facile fluorescence turn‐on assay for the sensitive detection of H2O2 and glucose is developed based on the target‐regulated redox state switch between Ce3+ and Ce4+. In this study, Ce3+ can efficiently quench the fluorescence of calcein while Ce4+ will not. Therefore, in the fluorescence‐quenched Ce3+/calcein‐based system, the H2O2‐catalyzed oxidation of Ce3+ to Ce4+ will lead to the fluorescence recovery of calcein, which can be recorded for the quantitation of H2O2. More interestingly, CeO2 nanoparticle, a fascinating material, exposes both Ce3+ and Ce4+ in a redox equilibration state on its surface. The H2O2‐regulated Ce3+/ Ce4+ redox state switch on CeO2 surface will be much easier and faster than that in the solution. Therefore, a CeO2/calcein complex‐based sensing system is further developed, and the detection limit of H2O2 can be pushed down to a low concentration of 8 nM, which is nearly three orders of magnitude lower than that of the Ce3+/calcein‐based sensing mechanism. Furthermore, by using the CeO2/calcein‐based system, glucose can be also sensitively detected with the assistance of glucose oxidase (GOx), and as low as 200 nM glucose can be clearly discriminated from the blank control. This proposed strategy provides a simple, cost‐effective, and highly sensitive approach for detecting H2O2, glucose as well as other oxidases‐related bioreactions, which may serve as a powerful tool for biosensing and clinical diagnostics.