Soybean protein-phosphatidylcholine was used as the emulsifier to prepare fish oil nano-emulsion by high pressure homogenization technology. The effect of concentrations of soybean protein, phosphatidylcholine and fish oil, and homogenous pressure on the average particle size, PDI, ζ-potential, turbidity of fish oil nano-emulsion were studied. Results with the highest desirability (the mean droplet size was (245±3.1)nm, PDI was 0.226±0.019, ζ-potential was (-30.2±0.6)mV and turbidity was (2.413±34.7)cm-1 was obtained with mass ratio of soybean protein content of 2%，phosphatidylcholine content of 0.2%, fish oil content of 1.5%, and homogenization pressure of 100MPa.Through the super high-resolution microscopy it was observed that fish oil was embedded in the composite emulsifier and evenly distributed in the emulsion system. Through the stability study of the highest desirability soybean protein-phosphatidylcholine fish oil nano-emulsion, it was found that nano-emulsion was remained stable against coalescence and phase separation for a period of 30d (storage period) at 4℃ and 25℃, respectively. The ability of the soybean protein-phosphatidylcholine fish oil nano-emulsion was stronger than that of Tween20 fish oil nano-emulsion. The highest desirability soybean protein-phosphatidylcholine fish oil nano-emulsion had good ionic resistance ability, and the influence of Ca2+ on stability of nano-emulsions was greater than Na+. In addition, the nano-emulsion was stable under all processing conditions that encountered during food processing at pH value of 7.0~10.0. The developed fish oil nano-emulsion had potential applications in the food industry.