Extracellular vesicles (EVs) have become a focal point in life sciences due to their roles in health and disease, with applications in diagnosis, drug delivery, and therapy. However, their heterogeneity and complex origins pose challenges in preparation and clinical use. This review discusses EV biogenesis, composition, and sources, focusing on advanced techniques for preparation and analysis, including microfluidic and non-microfluidic platforms, artificial intelligence, and electrochemical sensors. It highlights current challenges and future research directions. EVs are categorized into exosomes (40-200 nm), microvesicles (200-1000 nm), and apoptotic bodies (500-2000 nm), formed through different cellular processes. EVs contain proteins, nucleic acids, lipids, and metabolites, serving as biomarkers for disease diagnosis and therapeutic agents. EVs are sourced from various biological fluids, tissues, and cell cultures, with recent interest in non-human sources like cow's milk, plants, and bacteria for cost-effective production. EV preparation techniques include ultracentrifugation, density gradient centrifugation, size-exclusion chromatography, ultrafiltration, polymer precipitation, and microfluidics. Each method has advantages and limitations, with the choice depending on the research goals. Advanced methods, such as microfluidic devices and nanotechnology, offer improved efficiency and specificity in EV isolation. Affinity-based methods using antibodies, aptamers, lipids, peptides, and glycans enable targeted EV purification. Recent innovations include microfluidic chips with nanopatterns, magnetic beads, and bioorthogonal click chemistry for efficient EV capture and analysis. These advancements aim to enhance EV preparation and analysis, unlocking their biomedical potential for clinical applications.Extracellular vesicles (EVs) have become a focal point in life sciences due to their roles in health and disease, with applications in diagnosis, drug delivery, and therapy. However, their heterogeneity and complex origins pose challenges in preparation and clinical use. This review discusses EV biogenesis, composition, and sources, focusing on advanced techniques for preparation and analysis, including microfluidic and non-microfluidic platforms, artificial intelligence, and electrochemical sensors. It highlights current challenges and future research directions. EVs are categorized into exosomes (40-200 nm), microvesicles (200-1000 nm), and apoptotic bodies (500-2000 nm), formed through different cellular processes. EVs contain proteins, nucleic acids, lipids, and metabolites, serving as biomarkers for disease diagnosis and therapeutic agents. EVs are sourced from various biological fluids, tissues, and cell cultures, with recent interest in non-human sources like cow's milk, plants, and bacteria for cost-effective production. EV preparation techniques include ultracentrifugation, density gradient centrifugation, size-exclusion chromatography, ultrafiltration, polymer precipitation, and microfluidics. Each method has advantages and limitations, with the choice depending on the research goals. Advanced methods, such as microfluidic devices and nanotechnology, offer improved efficiency and specificity in EV isolation. Affinity-based methods using antibodies, aptamers, lipids, peptides, and glycans enable targeted EV purification. Recent innovations include microfluidic chips with nanopatterns, magnetic beads, and bioorthogonal click chemistry for efficient EV capture and analysis. These advancements aim to enhance EV preparation and analysis, unlocking their biomedical potential for clinical applications.