Extracellular vesicles (EVs) are lipid membrane-enclosed particles released by all cells and can be isolated from various sources, even from solid tissues. This study focuses on isolating and characterizing EVs from mouse lymph nodes (LNs). Male C57BL/6 mice were injected with complete Freund’s adjuvant, with or without ovalbumin. Inguinal and popliteal LNs were incised 9 days after immunization, and EV isolation was carried out using a combination of differential centrifugation and size-exclusion chromatography. The characteristic morphology of small and large EVs was confirmed by transmission electron microscopy. Particle size distribution and concentration were determined by nanoparticle tracking analysis, while protein and lipid contents were measured by bicinchoninic acid assay, and sulfo-phospho-vanillin assays, respectively, to calculate the protein-to-lipid ratio. Immune and EV markers were analyzed by using flow cytometry and Western blot assay, revealing significant changes between immunized mice compared to controls. This study establishes a novel protocol for isolating and characterizing EVs from LNs and highlights the impact of immunization on EV properties, offering insights into their roles in immune processes.