Alterations of cellular redox status are closely associated with physiological and pathological processes. Glutathione (GSH) and H₂O₂ should be the most representative redox couple in living cells. However, up to now, there is no way to reversibly detect GSH/H₂O₂. In this report, a near-infrared (NIR) fluorescent probe (Cy-O-Eb) for monitoring the changes of GSH/H₂O₂ levels in vivo was developed based on switching on–off a five-membered ring involved in ebselen. This probe could reversibly respond to GSH and H₂O₂ with high selectivity, sensitivity and mitochondrial targeting. It was successfully used to monitor the changes of redox status during apoptosis and the H₂O₂ changes at the wound margin in zebrafish larvae. Thus, the probe would provide an ideal tool for monitoring redox status changes and studying molecular events involved in redox regulation.