As the organs responsible for toxin transformation and excretion in the body, damage to the liver and kidneys induced by inevitable drug toxicity is the main cause of acute liver and kidney injury. P-Acetamidophenol overdose leads hypochlorous acid (HClO) to accumulate in the mitochondria of tissues, ultimately resulting in acute liver and kidney injury in humans, despite its clinical use as an antipyretic medicine. Herein, we report an HClO-activatable self-assembling ratiometric nanoprobe NRH-800-PEG for screening the upregulation of HClO by colocalization in mitochondria while monitoring the changes in the endogenous HClO levels in cells with ratiometric signals. Furthermore, NRH-800-PEG was constructed to evaluate injury by fluorescence ratio imaging in the tissues of inflammatory mice. Our strategy offers a novel tool for assessing disease progression during drug-induced liver and kidney injury.