Mitochondrial damage may lead to uncontrolled oxidative stress and massive apoptosis, and thus plays a pivotal role in the pathological processes of myocardial ischemia-reperfusion (I/R) injury. However, it is difficult for the drugs such as puerarin (PUE) to reach the mitochondrial lesion due to lack of targeting ability, which seriously affects the expected efficacy of drug therapy for myocardial I/R injury. We prepared triphenylphosphonium (TPP) cations and ischemic myocardium-targeting peptide (IMTP) co-modified puerarin-loaded liposomes (PUE@T/I-L), which effectively deliver the drug to mitochondria and improve the effectiveness of PUE in reducing myocardial I/R injury. In vitro test results showed that PUE@T/I-L had sustained release and excellent hemocompatibility. Fluorescence test results showed that TPP cations and IMTP double-modified liposomes (T/I-L) enhanced the intracellular uptake, escaped lysosomal capture and promoted drug targeting into the mitochondria. Notably, PUE@T/I-L inhibited the opening of the mitochondrial permeability transition pore, reduced intracellular reactive oxygen species (ROS) levels and increased superoxide dismutase (SOD) levels, thereby decreasing the percentage of Hoechst-positive cells and improving the survival of hypoxia-reoxygenated (H/R)-injured H9c2 cells. In a mouse myocardial I/R injury model, PUE@T/I-L showed a significant myocardial protective effect against myocardial I/R injury by protecting mitochondrial integrity, reducing myocardial apoptosis and decreasing infarct size. This drug delivery system exhibited excellent mitochondrial targeting and reduction of myocardial apoptosis, which endowed it with good potential extension value in the precise treatment of myocardial I/R injury.