Drug-induced liver injury (DILI) is a serious concern in modern medicine due to its unpredictability.Currently, biochemical serum markers are being used in DILI detection. However, these biomarkerbased methods lack sensitivity and specificity. A high intracellular level of hydroxyl radicals (cOH) has been regarded as an early indicator of DILI. Therefore, we proposed an cOH-responsive and hepatocyte-targeted nanoprobe via conjugation of carboxyfluorescein-labeled DNA and pegylated galactose on the surface of gold nanoparticles. The nanoprobe could bind to a hepatocyte-specific asialoglycoprotein receptor through galactose, and it could be internalized into liver cells. In the presence of high levels of cOH in DILI, the DNA could be cleaved to release carboxyfluorescein, leading to remarkable fluorescence enhancement for cOH detection. Confocal fluorescence imaging demonstrated that the nanoprobe could be successfully applied in monitoring high cOH levels resulting from acetaminophen or triptolide-induced liver injury, which may provide a simple but powerful protocol for the prediction of DILI.