Carotenoids are colored bioactive substances increasingly used due to their antioxidant properties, vitamin A precursor role, and ability to function as a natural food color. Knowledge of carotenoid behavior during high-heat processing and subsequent storage in emulsified food matrix is essential to expand their application natural food colors and neutraceuticals. Firstly, the physical, thermal, and colloidal stability of emulsions constructed from octenyl succinic anhydride-modified starch (OSA starch)-chitosan multilayered interfaces were investigated. Results of charge reversal from −32.4 ± 1.9 mV to +38.0 ± 0.8 mV indicate that multilayered interfaces were formed in emulsions. As measured by Z-average size, the emulsions were stable after the thermal treatment at 121 °C for 60 min, thus demonstrating a novel heat-stable multilayered emulsion. Subsequently, a select multilayered emulsion was loaded with β-carotene, and its storage stability was assessed. The degradation of β-carotene in an oil-in-water emulsion was better described with zeroth order kinetics; β-carotene dissolved in bulk oil was better described using a second-order kinetic equation. The presence of an encapsulating material around the oil droplets loaded with β-carotene enhanced its stability, which makes it instrumental in extending shelf-life and maintaining a consistent appearance. The results can be used to predict the availability of β-carotene during storage.