Hydrogen peroxide (HO) plays essential roles in redox signaling and oxidative stress, and its dynamic concentration is critical to human health and diseases. Here we report the design, syntheses, and biological applications of and for quantitative measurement of HO. Both probes were successfully applied to detect endogenous HO fluxes in living cells or zebrafish, and biological effects of multiple stress inducers including rotenone, arsenic trioxide, and starvation were investigated. As HO is a common by-product for oxidase oxidation, a general assay was developed for ultrasensitive detection of various metabolites (glucose, uric acid, and sarcosine). Moreover, cellular HO measurements were achieved for the first time by combining flow cytometry with live cell calibration. This study provides a pair of unique molecular tools for advanced HO bio-imaging and assay development.