Vitamin B, or riboflavin, is essential for maintaining healthy cellular metabolism and function. However, its light sensitivity, poor water solubility, and gastrointestinal barriers limit its storage, delivery, and absorption. Selecting suitable nanomaterials for encapsulating vitamin B is crucial to overcoming these challenges. This study employed chitosan-coated poly(lactic-co-glycolic acid) nanoparticles (CS-PLGA NPs) as a novel delivery system to enhance the bioavailability of vitamin B for food fortification and nutraceutical applications. The nanoparticles, with sizes below 200 nm, exhibited greater stability than PLGA NPs after freeze-drying and in simulated body fluids. Encapsulation improved the photostability of vitamin B under ultraviolet light and prolonged its release in simulated body fluids compared to non-encapsulated vitamin B. Furthermore, CS-PLGA NPs demonstrated higher uptake in intestinal epithelial cells (Caco-2), indicating enhanced transport and potential for use in fortified food systems. These findings underscore the promise of CS-PLGA NPs for delivering vitamin B in food, nutraceutical, and pharmaceutical applications. PRACTICAL APPLICATION: The use of chitosan-coated PLGA NPs for encapsulating vitamin B offers a promising solution to enhance its bioavailability, especially for individuals with gastrointestinal absorption issues. This formulation improves stability, controlled release, and cellular uptake, which can lead to more effective supplementation strategies in nutraceutical and pharmaceutical applications. It could benefit patients with vitamin B deficiencies, such as those with malabsorption disorders, by ensuring efficient delivery through the gastrointestinal tract. Additionally, this approach can be applied to other water-soluble vitamins or bioactive compounds, offering a versatile platform for improving the efficacy of oral supplements.