Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H₂O₂), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H₂O₂ during the early events of wound responses in zebrafish larvae expressing a genetically encoded H₂O₂ sensor. This reporter revealed a sustained rise in H₂O₂ concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100–200 μm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H₂O₂ pattern, and the first evidence that H₂O₂ signals to leukocytes in tissues, in addition to its known antiseptic role.