Abstract: In order to solve the bad flavor and easily oxidation of bitter gourd seed oil (BGSO), whey protein isolate, apple pectin and chitosan oligosaccharide were used as emulsifiers, and electrostatic layer-by-layer self-assembly technology was used to encapsulate BGSO to make BGSO maintained good physical stability. In this study, the preparation process of multilayer emulsion of BGSO was investigated, and the mean diameter, polydispersity index, ζ-potential, microstructure observation, rheological properties, physical stability and main fatty acid composition of BGSO multilayer emulsion were measured. The results showed that the three emulsions prepared when the homogenization pressure was 600 bar, homogenization time was 5 times, apple pectin concentration was 3.5% (w/v), secondary emulsion pH was 4.4, chitosan oligosaccharide concentration was 2% (w/v), and tertiary emulsion pH was 2.2, had smaller mean diameter, which were 460±3, 670±9 and 1623±10 nm, respectively, and had smaller polydispersity indexes were 0.073±0.004, 0.258±0.020 and 0.480±0.034 respectively, and higher ζ-potential absolute value, were −22.3±0.8, −19.6±0.9 and 16.3±0.3 mV respectively. And the microstructure observation results showed that the prepared three emulsion droplets were relatively dispersed, and the fatty acid composition measurement results indicated that the preparation process might cause the oxidation of unsaturated fatty acids in BGSO, the physical stability test results showed that the physical stability of BGSO double-layer and three-layer emulsion were better than single-layer emulsion. This study would provide a theoretical reference for improving the stability of BGSO and broadening the application of BGSO.