Fluorescein-labeled hyaluronic acids (HA) were immobilized on gold nanoparticles for reactive oxygen species (ROS) detection. The efficacy of HA immobilized gold nanoparticles (HHAuNPs) was evaluated in a stroke animal model. The stroke rat model was produced by transient middle cerebral artery occlusion (MCAO), which induced transient ischemia and reperfusion (I/R) in the brain. The increase of ROS in the I/R brain was confirmed by TBARS assay with the brain extracts. For brain imaging, HHAuNPs were injected into the rat brain 1 h before transient MCAO. Five hours after the injection, the rats were sacrificed and the brains were subjected to imaging analysis. The results showed that stronger signals were detected in the I/R brains than in the normal brains. To identify the time window for effective detection of ROS, HHAuNPs were injected into the post-ischemic rat brains at various time points. The results showed that ROS level reached a maximum at 24 h after the transient MCAO. Also, a live imaging study was performed with HHAuNPs in the normal and I/R animals. The results confirmed that ROS level increased in the I/R animal group with time, while the signal was decreased in the normal animal group. Together, our results suggest that HHAuNPs may be useful to monitor ROS level in the ischemic brain and to identify the infarct areas in ischemic brains for the treatment of stroke.