Typically, all the normal as well as cancer cells in the body secrete extracellular vesicles (EVs), with different types of cells secreting different types of EVs to induce specific biological effects in cells. In addition, EVs exhibit characteristics of high abundance and stability and are not easily degradable, rendering them candidate carriers for diagnostic and therapeutic clinical interventions in the future. However, the targeting efficacy and bioaccumulation of EVs require further evaluation in detail. Current studies predominantly employ antibodies for tracking the biodistribution of EVs. However, the antibodies are coated on the outer membrane of EVs, leading to interactions between the exposed protein regions and the biological molecules in the microenvironment, affecting the stability and distribution of EVs. The current study employs persistent luminescence nanoparticles (PLNs) emitting in the near-infrared (NIR) region for tracking the biodistribution of EVs over an extended duration. Excitation of PLNs can be accomplished in vitro using ultraviolet light. The NIR photobiological imaging 1−9 h following the intravenous injection of the PLNs in mice provides a diagnostic system for long-term in situ tumor tracking. The synthesis of PLNs involves the use of mesoporous silica nanoparticles as a template; the high stability and uniform particle size of the PLNs enhances exocytosis in cells subjected to the electrochemical technology of Transwell cell electroporation (TCE). The enhanced exocytosis promotes the secretion of numerous PLN-embedded EVs (PLN@EVs) into the culture medium, which are subsequently purified by ultracentrifugation. PLN@EVs can be employed not just as a diagnostic tool but also as a tumor-homing platform; production of large quantities of the long-term traceable PLN@EVs and their targeting to the tumor tissues such as nonsmall cell lung cancer would greatly facilitate the diagnosis of cancer.