Milk extracellular vesicles (EVs) represent promising drug delivery platforms, yet current isolation methods face scalability challenges. Ultracentrifugation (UC), the gold standard, is expensive and energy-intensive, limiting pharmaceutical and pharmacological implementation. This study aimed to standardize scalable EV isolation methods and evaluate cheesemaking whey as a sustainable alternative to milk for therapeutic applications. We systematically compared isolation techniques including UC, tangential flow filtration (TFF), and polyethylene glycol (PEG) co-precipitation across milk and whey from bovine, caprine, and ovine sources. Casein removal strategies using acidification and EDTA treatment were evaluated. Functional validation included in vitro simulated gastrointestinal digestion and in vivo pharmacokinetic studies of miRNA-loaded EVs in murine models. Our results demonstrate that cheesemaking whey serves as a sustainable EV source with reduced casein contamination and comparable yields to milk. Both TFF and PEG co-precipitation effectively concentrate EVs from whey, offering viable UC alternatives for pharmaceutical scale-up, though additional purification steps are required for optimal purity. Acidification effectively removes protein contaminants from milk but shows minimal benefits for whey. Cow whey provided the highest EV yields among species evaluated. Functionally, EV-encapsulated miRNAs demonstrated superior gastrointestinal stability compared to free miRNAs and exhibited enhanced bioavailability in multiple target tissues including cardiac, splenic, and skeletal muscle following oral administration. These findings establish cheesemaking whey as a sustainable, pharmaceutical-grade EV source and validate scalable isolation methods suitable for therapeutic applications, providing essential groundwork for clinical translation of milk-derived EV-based drug delivery systems. © 2025 Elsevier B.V., All rights reserved.