Abstract Hypobromous acid (HOBr) has been implicated in many physiological and pathological conditions. Therefore, real-time monitoring of HOBr fluctuations in biosystem plays a key role for understanding pathophysiological processes. To date, it remains a challenge to design fluorescent probes specific toward HOBr, because HOBr and HOCl have similar chemical properties and the former has a relatively lower concentration in comparison with the former in living system. Herein, a Forster resonance energy transfer (FRET)-based ratiometric fluorescent HOBr probe (Cou-RhB) was developed. The probe consists of a coumarin donor and a rhodamine acceptor. Upon treatment of Cou-RhB with HOBr, the electrophilic bromination of xanthene ring occurs, which shifts the equilibrium of rhodamine from the highly absorbing and fluorescent zwitterion form to the colorless and non-fluorescent spirolactone form, thus decreasing the FRET efficiency within the probe. The above reaction affords a large emission wavelength shift (ca. 89 nm), and ratiometric sensing of HOBr can be realized by measuring the ratio of coumarin- to rhodamine-type intensities (I491/I580). Cou-RhB responds to HOBr with a fast kinetics (∼ 10 s), high sensitivity and excellent specificity and has been applied for ratiometric imaging of HOBr inside live cells and zebrafish.