The emulsion and hydrolysate obtained from enzymatic hydrolysis of Jatropha curcas L. seeds were used as compound emulsifier, and the enzymatic hydrolysate nanoemulsion of Jatropha curcas L. seeds was prepared by high pressure homogenization technology. The effects of additive amount of emulsion and hydrolysate in the enzymatic product of Jatropha curcas L. seeds, as well as high pressure homogenization pressure and frequency on the average particle size, polydispersity index (PDI),ζ-potential, turbidity, emulsion productive rate and TSI of nanoemulsion were studied, results with the highest desirability of preparing the nanoemulsion were obtained with the additive amount of emulsion at 15.55%, the additive amount of hydrolysate at 45.25%, high pressure homogenization pressure at 91MPa and homogenization frequency at four times. Upon these conditions, the average particle size was 297.2nm, turbiscan stability index (TSI) was 2.98 and the emulsion productive rate was as high as 92.47%. The enzymatic hydrolysate nanoemulsion of Jatropha curcas L. seeds was finally prepared, it was observed through the super highresolution microscopy that the particles of it were uniform in the size and evenly distributed in the emulsion system. In addition, it was found that the interfacial protein content of the nanoemulsion was as high as 31.20mg/m2.