Abstract: Saccharina japonica is a commercially important brown alga with significant potential for high-value utilization in the blue economy. Extracellular vesicles derived from S. japonica (SJEVs) may carry marine-specific bioactive molecules, yet their isolation and characterization remain unexplored. This study aims to establish and compare efficient isolation methods for SJEVs and evaluate their physicochemical properties and biosafety to support downstream applications. SJEVs were isolated from rehydrated dried kelp using three methods: a commercial kit (BL920A), polyethylene glycol (PEG6 000) precipitation (8%, 10%, 12%, 16%), and chitosan precipitation (50, 100, 150, 200 μg/mL). The isolated SJEVs were comprehensively characterized using transmission electron microscopy, dynamic light scattering for particle size and Zeta potential, and biochemical assays for protein, polysaccharide, RNA, and DNA content. Biosafety was assessed via CCK-8 cytotoxicity assay on L-929 cells and hemolysis assay using rabbit erythrocytes. All methods successfully yielded SJEVs with typical bilayer vesicular morphology. Mean particle sizes ranged from (137.42±6.09) nm (chitosan 100 μg/mL) to (227.49±8.18) nm (chitosan 200 μg/mL), with the kit method giving (153.36±1.45) nm. Zeta potentials ranged from -33.97 mV (chitosan 100 μg/mL) to -25.76 mV (12% PEG), indicating good colloidal stability. Protein content was highest in kit-isolated SJEVs (31.1±1.2) μg/mL, followed by chitosan 200 μg/mL (29.83±1.29) μg/mL and 10% PEG (28.23±1.40) μg/mL). Polysaccharide content ranged from (121.22±5.45) μg/mL (chitosan 50 μg/mL) to (157.87±31.25) μg/mL (12% PEG). RNA purity was optimal in chitosan 150 μg/mL (A260/A280 = 2.08±0.20), significantly higher than that of the kit method (1.00±0.09). DNA concentration peaked in chitosan 200 μg/mL (6.50±0.30) ng/mL) but with very low purity (A260/A280 = 0.37±0.04), suggesting co-precipitation of free DNA. Cytotoxicity assays showed >85% cell viability across all tested concentrations (10-20 000 μg/L) of all three selected SJEVs, and hemolysis rates remained below 5% for all tested SJEVs up to 30,000 μg/L, confirming excellent biosafety. All three methods effectively isolate SJEVs with intact structure and good biosafety. The 10% PEG and 100 μg/mL chitosan offer balanced performance in yield, purity, and scalability, making them suitable for large-scale production. The strong negative surface charge of SJEVs, likely due to sulfated polysaccharides, enhances stability and biointerface interaction potential. This study establishes the first methodological framework for isolating SJEVs from a major brown alga, supporting their development as marine-derived nanocarriers for drug delivery, functional foods, and green sustainable agriculture, thereby contributing to the sustainable development of the blue economy and aquatic industry. Future work should focus on scaling up production, identifying marine-specific functional molecules, and validating in vivo efficacy.