The purpose of this study is to produce an oil-in-water nano-emulsion by ultrasonication. Casein combined with inulin used as continuous phase, while dispersed phase consisted of fish oil. The size of the nano-emulsion and the pH of nano-emulsion were characterized. Ultrasound has been used for preparing emulsion by 24 KHz intensity for 120 seconds. Prepared nano-emulsion was investigated by particle matrix. Result shows that ultrasonication is an efficient emulsification technique producing small emulsion droplets. Nano-emulsions were very small droplet size (<172.344 nm) .In the present work, our aim was to evaluate kind of continuous phase materials and its concentration on the pH of the Oil/water nano-emulsions and the size distribution of nano-emulsion droplet. Considering the difference in structure in our study shows the mean particle diameter of nano-emulsion droplets decreased with increasing fish oil concentration. This research proved that type and density of used components as continuous has effect on the properties of nano-emulsion. The ultrasonic technology used in this study could lead to application in the food industry.