The variations in average particle size, zeta potential, free fatty acids (FFA) release rate, and the bioavailability of menthol under in vitro simulated digestion conditions of peppermint oil nanoemulsion were investigated. 3D confocal laser scanning microscopy and Cryo-scanning electron microscopy were used to observe the microstructure characteristics of peppermint oil nanoemulsion, which indicated that soybean protein was completely adsorbed at the oil-water interface of the nanoemulsion and presented a core shell structure. And the results indicated that FFA release rate and menthol bioavailability of peppermint oil nanoemulsion prepared by using high-pressure homogenization were much higher. In the simulated gastric digestion phase, the average particle size and the zeta potential of the nanoemulsion increased, and droplet polymerization appeared. After the simulated intestinal, the interfacial protein of nanoemulsion was hydrolyzed, and the oil droplets were digested, which resulted in the decreased particle size and increased absolute value of zeta potential.