In view of the fact that perilla oil tends to be oxidized during storage, with soy protein isolate (SPI) or its blends with chitosan (CS) and sodium alginate (SA) as the emulsifier, electrostatic layer-by-layer self-assembly was used to encapsulate perilla oil to maintain its physical stability and for its sustained release. The microscopic morphology and stability of the perilla oil single-layer emulsion, double-layer emulsion and three-layer emulsion were investigated, and an in vitro simulated digestion model was established to determine changes in the fatty acid composition of the three emulsions before and after digestion by gas chromatography. The results showed that the three emulsions, prepared using a wall material composed of 1.0% SPI solution, 2.0% CS solution, and 1.5% SA solution, had small particle size, high potential, and good physical and chemical stability. Under acidic conditions, the multi-layer emulsions could better protect polyunsaturated fatty acids. As the number of layers increased, the oxidation rate of perilla oil became slower. The in vitro simulated digestion results showed that the three-layer emulsion had a better sustained-release effect than the single-layer and double-layer emulsions, and the multi-layer emulsion could ensure the effective release of fatty acids from the oil. These findings can guide the development of slow-release systems for oils.