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Investigation was made on the effects of the peppermint oil nanoemulsion on the average particle size, zeta potential, turbidity, FFA release rate and the bioavailability of menthol in vitro simulated digestion process. It was found that peppermint oil was completely packed by SPI and nanoemulsion droplet was in the spherical shape by using confocal laser scanning 3D microscopy to observe the microstructure of peppermint oil nanoemulsion, indicating that the soybean protein was completely adsorbed at the oil-water interface of the nanoemulsion and presented a core shell structure. The results indicated that the FFA release rate and menthol bioavailability of the peppermint oil nanoemulsion prepared by high-pressure-homogenization were much higher than that in the control group of peppermint oil. In the simulated gastric digestion phase, the average particle size and the zeta potential of the nanoemulsion were increased, while the turbidity was decreased and the microstructure of the digestive emulsion showed the phenomenon of droplet polymerization. After the simulated intestinal juice was digested, the interfacial protein of peppermint oil nanoemulsion was hydrolyzed, the oil droplets were digested, the average particle size of the emulsion was decreased, and the absolute value of zeta-potential was increased. The stability of the peppermint oil nanoemulsion was checked by the Turbiscan stability analyzer, and the stability index was found to be 2.8. There was no emulsion floatation and flocculation occurred during the test.