Hydrogen peroxide (HO) is a key redox intermediate generated within cells. Existing probes for HO have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric HO probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of HO diffusion from the mitochondrial matrix, to find a functional output of HO gradients in polarized cells, and to prove the existence of HO gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time HO imaging in various biological contexts.