Allyl isothiocyanate (AITC), an organosulfur compound in cruciferous vegetables, is a natural antimicrobial and potential chemopreventive agent. However, the instability of AITC in aqueous systems restrains its applications. In this study, oil-in-water AITC nanoemulsion was prepared by the emulsion inversion point (EIP) method, aiming at improving the aqueous stability of AITC. The optimal hydrophilic-lipophilic balance (HLB(op)) value of surfactants containing Tween 80 and Span 80 was established at 11.0-13.0, yielding nanodroplets with diameters of 137-215 nm. The mechanism of droplet formation within the HLP(op) region was discussed in terms of the possible structure of adsorbed surfactant layers at the oil-water interface in multiple emulsion droplets. In a 6.5-month storage test, the droplet sizes and the count rates (intensity of scattered light) of nanoemulsions decreased only slightly by 4-13% (depending on surfactant-to-oil ratio), even in highly diluted status, indicating the desirable stability of the nanoemulsions. Moreover, the nanoemulsion demonstrated superior protection against AITC degradation (78% remaining after 60 d at 30 °C), compared with protein nanoparticles as well as non-encapsulated aqueous dispersion. This work shows for the first time that AITC can be formulated into nanoemulsions and thus obtains satisfactory aqueous solubility and chemical stability.