Determination of hydroxyl radical ((•)OH) with high sensitivity and accuracy in live cells is a challenge for evaluating the role that (•)OH plays in the physiological and pathological processes. In this work, a ratiometric fluorescence biosensor for (•)OH was developed, in which gold nanocluster (AuNC) protected by bovine serum albumin was employed as a reference fluorophore and the organic molecule 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) acted as both the response signal and specific recognition element for (•)OH. In the absence of (•)OH, only one emission peak at 637 nm ascribed to AuNCs was observed, because HPF was almost nonfluorescent. However, fluorescence emission at 515 nm attributed to the HPF product after reaction with (•)OH--dianionic fluorescein--gradually increased with the continuous addition of (•)OH, while the emission at 637 nm stays constant, resulting in a ratiometric determination of (•)OH. The developed fluorescent sensor exhibited high selectivity for (•)OH over other reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, and other biological species, as well as high accuracy and sensitivity with low detection limit to ∼0.68 μM, which fulfills the requirements for detection of (•)OH in a biological system. In addition, the AuNC-based inorganic-organic probe showed long-term stability against light illumination and pH, good cell permeability, and low cytotoxicity. As a result, the present ratiometric sensor was successfully used for bioimaging and monitoring of (•)OH changes in live cells upon oxidative stress.