Genetically encoded probes monitoring H 2 O 2  fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H 2 O 2  caused by extracellular peroxides; expression of human aquaporin 8 channels H 2 O 2  entry into fission yeast decreasing membrane gradients. The probe also detects H 2 O 2  bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H 2 O 2  in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H 2 O 2  fluctuations.