This review provides an overview of the biogenesis, composition, and sources of Extracellular Vesicles (EVs), emphasizing their critical roles in health and disease. It discusses the challenges in EV preparation and analysis due to their heterogeneity and complex origins. The review highlights contemporary techniques for EV preparation, including microfluidic and non-microfluidic platforms, and innovative approaches that incorporate artificial intelligence and electrochemical sensors. It also covers advanced methods such as DNA nanotechnology and peptide-based methods for EV isolation. The review identifies current challenges and outlines future research avenues, aiming to motivate researchers to innovate and expand methods for EV preparation and analysis to fully unlock their biomedical potential.This review provides an overview of the biogenesis, composition, and sources of Extracellular Vesicles (EVs), emphasizing their critical roles in health and disease. It discusses the challenges in EV preparation and analysis due to their heterogeneity and complex origins. The review highlights contemporary techniques for EV preparation, including microfluidic and non-microfluidic platforms, and innovative approaches that incorporate artificial intelligence and electrochemical sensors. It also covers advanced methods such as DNA nanotechnology and peptide-based methods for EV isolation. The review identifies current challenges and outlines future research avenues, aiming to motivate researchers to innovate and expand methods for EV preparation and analysis to fully unlock their biomedical potential.