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Many studies have shown that grape seed oil (GSO) is one of the vegetable fats that are plentiful in essential fatty acids and can be used as a fat substitute or to modify fat in food products to reduce saturated fatty acids. However, due to its low solubility and high sensitivity to oxidation, it is necessary to develop delivery systems that can distribute GSO in food more effectively. Recently, the preparation of emulsions using the layer-by-layer (LBL) method has many advantages in delivering lipid-soluble functional compounds. This research was used to check the formation of GSO oil-loaded primary, secondary and tertiary multilayer emulsions stabilized by mixture of anionic gelatin, cationic chitosan, and anionic basil seed gum (BSG) as the aqueous phase at pH 5, prepared using a layer-by-layer electrostatic deposition technique. Multilayer emulsions prepared by GSO and a mixture of gelatin, chitosan, and BSG as the aqueous phase at pH 5. Finally, the effect of the number of layers on the physicochemical properties (particle size, viscosity, turbidity, refractive index, and physical stability) and oxidative stability (peroxide value, thiobarbituric acid value, and fatty acid profile) during the storage time (30 days) at two temperatures 25 °C & 4 °C was investigated. Also, the zeta potential and Fourier transform infrared spectroscopy (FTIR) of mono-layer and multi-layer emulsions were investigated. The results revealed that by increasing the number of layers of multi-layer emulsion of GSO, the stability has improved. Thus, the tertiary emulsion has been more effective than the other two emulsions in maintaining the physicochemical characteristics and stability over time (P < 0.001). Morphological characterization and FTIR spectroscopy results confirmed that gelatin, chitosan, and BSG were successfully loaded into the LBL emulsions. This study can improve the original percept of multilayer emulsions and promulgate their potential applications for the entire encapsulation of essential fatty acids to enrich and prevent peroxide attack., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)