AbstractRetinal ischemia–reperfusion (RIR) injury is involved in the pathogenesis of various vision‐threatening diseases. The overproduction of reactive oxygen species (ROS) is thought to be the main cause of RIR injury. A variety of natural products, including quercetin (Que), exhibit potent antioxidant activity. However, the lack of an efficient delivery system for hydrophobic Que and the presence of various intraocular barriers limit the effective retinal delivery of Que in clinical settings. In this study, we encapsulated Que into ROS‐responsive mitochondria‐targeted liposomes (abbreviated to Que@TPP‐ROS‐Lips) to achieve the sustained delivery of Que to the retina. The intracellular uptake, lysosome escape ability, and mitochondria targeting ability of Que@TPP‐ROS‐Lips were evaluated in R28 retinal cells. Treating R28 cells with Que@TPP‐ROS‐Lips significantly ameliorated the decrease in ATP content, ROS generation, and increase in the release of lactate dehydrogenase in an in vitro oxygen–glucose deprivation (OGD) model of retinal ischemia. In a rat model, the intravitreal injection of Que@TPP‐ROS‐Lips 24 h after inducing retinal ischemia significantly enhanced retinal electrophysiological recovery and reduced neuroinflammation, oxidative stress, and apoptosis. Que@TPP‐ROS‐Lips were taken up by retina for at least 14 days after intravitreal administration. Molecular docking and functional biological experiments revealed that Que targets FOXO3A to inhibit oxidative stress and inflammation. Que@TPP‐ROS‐Lips also partially inhibited the p38 MAPK signaling pathway, which contributes to oxidative stress and inflammation. In conclusion, our new platform for ROS‐responsive and mitochondria‐targeted drug release shows promise for the treatment of RIR injury and promotes the clinical application of hydrophobic natural products.