Recently, attention on plant extracellular vesicles (EVs) has increased alongside growing demands for standardizing their extraction methods. In this study, we compared three methods for isolating EVs from plants, including a novel Enzyme method (E method) designed for rapid apoplastic EV extraction. The E method involves macerozyme-mediated apoplastic washing fluid (AWF) collection followed by EV isolation using polymers and size-fractioning membranes. Compared to the freeze-grinding method (F method) and syringe-infiltration-ultracentrifugation method (SI method), the E method yielded more EVs sized between 50–150 nm and showed comparable purity based on marker protein analysis. Additionally, it facilitated higher EV yield and shorter isolation times, making it suitable for both adult leaf and seedling samples. Assessment in mutant backgrounds (patl1, syp61, and pen1) confirmed the method's robustness across different genetic contexts. These findings highlight the E method as an efficient and reliable approach for plant EV isolation and characterization.