Myocardial dysfunction caused by cardiomyocyte apoptosis under ischemic and hypoxic conditions is the pathological basis of most cardiovascular diseases. Current diagnosis of myocardial dysfunction still focuses on the symptomatic stage, usually after the occurrence of the irreversible remodelling and functional impairment. Thus, early stage identification of the apoptotic cardiomyocytes induced by hypoxia is highly significant for preventing the onset and delaying the progression of myocardial dysfunction. Herein, a novel Au-Se nanoprobe with strong anti-interference capability was developed for simultaneous real-time in situ monitoring the expression of Lon protease (Lon) and Caspase-3 with high-fidelity in living cardiomyocytes. As Lon upregulation plays a major role in the initiation of hypoxia-induced apoptosis and Caspase-3 is a marker protein for apoptosis, the nanoprobe has been successfully applied for imaging the activation of Lon-Caspase-3 apoptotic signalling pathway and assessing the state of cardiomyocytes under hypoxic conditions. Furthermore, combining with mitochondrial HO probe-MitoPY1, the nanoprobe was also used to confirm the synergistic effect of Lon and ROS on hypoxia-induced apoptosis of cardiomyocytes and evaluate the function of ROS scavenger on attenuating such apoptosis. This work proposed a promising strategy for early diagnosis, prevention and treatment of hypoxic-ischemic myocardial dysfunction.Copyright © 2021 Elsevier B.V. All rights reserved.