There is a strong medical need for precision imaging of ischemic tissue and even more so for delivery of drugs specifically to the ischemic tissue at risk of functional damage. To date, no targeting epitopes or mechanisms have been established in ischemia/reperfusion injury that allow specific molecular targeting for either diagnosis or therapy, a deficiency that contributes to myocardial infarction being the leading cause of mortality and a major contributor to morbidity worldwide. Reactive oxygen species (ROS) are locally increased at the site of ischemia/reperfusion injury. Utilizing these as a unique molecular targeting mechanism in developing ROS‐responsive nanoparticles, this study illustrates unique designed ROS‐responsive, self‐assembled fluorescent nanoparticles as tools for diagnostic and therapeutic targeting of tissue that is undergoing ischemia/reperfusion injury. This ROS‐responsive fluorescent nanoprobe has been evaluated in a mouse model of myocardial infarction, applying 1 h of ischemia via temporary ligation of the left anterior descending coronary artery and measuring fluorescence signals after reperfusion. This study shows highly specific targeting to the ischemic/reperfused myocardium throughout the first 24 h post reperfusion. Overall, this study identifies ROS‐responsive fluorescent nanoparticles as an ideal platform for the localized delivery of imaging agents and novel therapeutics to the infarcted myocardium.