Functional oils have broad application prospects in functional foods and beverages because of their rich beneficial ingredients and healthier intake. The small droplets of the nanoemulsion enhance the effective delivery, solubility and bioavailability of the various hydrophobic food components. This study used a mixed oil phase of green tea seed oil and fractionated coconut oil, compared the emulsifying properties of natural surfactants: Whey protein isolate, soy lecithin, tea saponin and synthetic surfactant: Tween 80 in the preparation of nanoemulsions by ultrasonic method. In particular, the impact of emulsifier type and concentration, pH, ionic strength, and heat treatment on the mean particle size and ξ-potential were investigated. The long-term storage stability of the fabricated nanoemulsions was also monitored during storage at different temperatures. In addition, the effects of emulsifier type on the bioavailability of nanoemulsions were evaluated. For all nanoemulsions studied, the mean particle size decreasing with increasing emulsifier concentration. Tea saponin and soy lecithin can produce smaller droplets of nanoemulsion than Whey protein isolate. Tea saponin has the same emulsifying ability as Tween 80. Presumably tea saponin-stabilized droplets may be maintained by electrostatic repulsion and steric repulsion. All of the nanoemulsions significantly improved the bioavailability of the mixed oil phase compared to the unemulsified oil phase. This study highlights the potential of natural surfactants in the ultrasonic preparation of nanoemulsions containing functional oils, and provides a basis for the application of natural surfactants and new functional oils in food industry.