Extracellular vesicles (EVs) isolated from Aloe vera (L.) Barbadensis exhibit promising biomedical applications, yet standardized isolation protocols remain elusive. This study systematically evaluates three isolation methods—serial centrifugation (SC), Ultracentrifugation (UC), and Reverse Serial Microfiltration (RSM)—for their efficacy in yield, purity, and scalability. SC demonstrated the highest protein yield (2.53 ± 0.12 mg/mL; p < 0.01 vs. UC/RSM), while UC achieved superior vesicle homogeneity (142 ± 8 nm in diameter, polydispersity index 0.18 ± 0.02). RSM emerged as the most scalable method, reducing processing time by 67% (45 minutes) and costs by 60% compared to UC, with 85 ± 5% EV recovery. Contaminant analysis revealed SC-introduced non-vesicular proteins (12 ± 3%), whereas UC and RSM maintained >90% purity via TEM and nanoparticle tracking analysis. These results provide a pragmatic framework: UC for fundamental research, SC for high-yield applications, and RSM for industrial translation. The study addresses critical gaps in plant EV bioprocessing, offering actionable insights for therapeutic and commercial development.