Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 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 H2O2 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 H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts. Display omitted •HyPer7 is an ultrasensitive, ultrafast, and pH-stable indicator for H2O2•Being controlled by the Trx system, H2O2 does not diffuse out from mitochondria•Intensity of the H2O2 gradient associates with the stability of cellular protrusions•H2O2 gradients can be visualized in wounded tissues with HyPer7 Pak et al. have developed HyPer7, a next-generation genetically encoded fluorescent probe for H2O2 detection. Importantly, HyPer7 is resistant to pH changes. Applying HyPer7 to study H2O2 diffusion from the mitochondrial matrix, the authors decipher the topology of H2O2 production by Complex I and visualize oxidant gradients in cell motility and wounded tissue.