Reactive oxygen species (ROS) are largely produced under pathological situations. To understand the etiology of disease, it is urgent to develop efficacious probes for detecting ROS. Herein, a novel nanoconjugate detection system constructed from gold clusters (AuNCs) and quantum dots (QDs) for fluorescence ratiometric-sensing ROS was reported. Upon interacting with ROS, the red emission fluorescence (645 nm from QDs) in the detection system gradually decreased, while the green fluorescence (480 nm from AuNCs) changed little. The fluorescence ratio at the 2 wavelengths (I480 nm /I645 nm ) was linearly correlated with the ROS, which could be used for the real-time ratiometric detection of ROS. The developed nanoconjugates could be applied to monitor the ROS in inflammatory cells for its ability of generating abundant ROS and uptaking ability to nanoparticles. The stimulated ROS in inflammatory cells were monitored by AuNC-QD and the results were consistent with the traditional 2', 7'-dichlorofluorescin diacetate method, confirming the reliability of the developed method. Featured with the merits of higher photostability, low background, high accuracy of ratiometric detection, the AuNC-QD conjugate demonstrated its potential to be the probe for real-time ROS detection in inflammatory cells.