Abstract <p>Plant extracellular vesicles (exosomes) are considered promising nanostructures for biomedical and biotechnological applications; however, methods for their isolation from plant cell cultures remain insufficiently optimized. The aim of this study was to evaluate the efficiency of exosome isolation from callus and suspension cultures of <i>Vitis vinifera</i> and to investigate the effects of culture age and extraction method on the yield of extracellular vesicles. It was demonstrated that suspension culture provides a higher exosome yield compared to callus tissue. Isolated particles were characterized by a size of 90–120 nm and a negative zeta potential, which corresponded to typical properties of plant extracellular vesicles. The maximum exosome yield was observed with a two-week-old suspension culture, which corresponded to the phase of intense cell growth. Comparison of extraction methods revealed that mechanical homogenization using a blender was the most efficient approach for exosome isolation, providing a high particle yield without significant deterioration in sample purity. The results demonstrate the potential of <i>V. vinifera</i> suspension cultures as a scalable platform for the production of plant-derived exosomes and may be useful for the development of biotechnological systems for the isolation of plant extracellular vesicles.</p>