<p>Plant-derived extracellular vesicles (PDEVs) are transforming nanomedicine as sustainable, biocompatible nanocarriers. They facilitate intercellular communication and transport bioactive molecules, but clinical translation is hindered by inconsistent isolation protocols, unclear therapeutic mechanisms, limited cross-plant comparisons, and undefined cargo sorting. This review addresses these gaps by evaluating limitations in current isolation and characterization methods to improve yield, purity, and reproducibility. Key findings show that PDEVs’ cargos (proteins, lipids, miRNAs, and metabolites) drive anti-inflammatory, antioxidant, anticancer, and regenerative effects. These target diseases like inflammatory bowel disease, alcoholic liver disease, cancer, and COVID-19 via mechanisms such as cytokine modulation, ROS reduction, and apoptosis induction. Preclinical evidence from vesicles derived from ginger, grape, citrus, turmeric, and aloe supports this. As drug nano-delivery platforms, PDEVs enhance bioavailability and target tissues like the gastrointestinal tract and brain, offering low-toxicity alternatives to synthetic systems. This review provides a roadmap for standardized protocols and mechanistic insights through preclinical evidence, optimized isolation, and cross-kingdom communication. PDEVs represent an eco-friendly frontier in nanomedicine with strong potential for clinical and pharmaceutical applications.</p> Graphical Abstract <p>Graphical abstract illustrates the summary of isolation, characterization, and therapeutic applications of plant-derived extracellular vesicles (PDEVs). Enriched with bioactive molecules, PDEVs serve as nanocarriers for targeted drug delivery, showing potential in treating inflammatory and metabolic diseases including IBD, arthritis, alcoholic liver disease, and cancer.</p> <p></p>