Abstract: In order to prepare tea tree oil（TTO） nanoemulsion with high stability, and evaluate the quality of the prepared TTO nanoemulsion, in the experiment, TTO nanoemulsion was prepared by high-energy shear method, and the optimal formulation of TTO nanoemulsion was determined based on the pseudo-ternary phase diagram. Appearance and characterization of TTO nanoemulsion were observed by optimal formulation. Type identification was carried out by staining method and pH was determined using pH meter. The surface morphology was observed by transmission electron microscopy, and the particle size and potential were determined and examined for stability by a laser particle sizer; the minimum inhibitory concentration（MIC） of TTO nanoemulsion and TTO against Pasteurella and Salmonella. was determined by using the two fold dilution method. The results showed that the optimal formulation of TTO nanoemulsion was 12.95% castor oil polyoxyethylene ether（EL-40）, 6.5% polyethylene glycol 400（PEG-400）, 8.33% TTO, and 72.22% purified water. The TTO nanoemulsion prepared by using the optimal formulation was an oil-in-water（O/W） type emulsion with an average pH value of 8.32, an average particle size of 15.7 nm and a surface potential of（-4.29±0.26） mV. The TTO nanoemulsion was diluted 20-120 times with water and left to stand for 24 h without any delamination or precipitation. After centrifugation at 4 000, 6 000, 8 000, 10 000 r/min for 20 min, there was no change in the appearance and properties of the nanoemulsion, and there was no precipitation, delamination and other phenomena produced. After being treated at different temperatures（-20 ℃, 4 ℃, 25 ℃, 40 ℃, 60 ℃, 80 ℃） for 30 min, there was no obvious change in its appearance, and its appearance was more transparent after being heated at 60 ℃, and delamination appeared after being heated at 80 ℃. It was observed after 30 d at 4 ℃ without any abnormal changes. The TTO nanoemulsion was diluted 100 times, 1 000 times and 10 000 times with aqueous solutions of pH value 5, 6, 7, 8 and 9, respectively, and then observed after 24 h of standing, and there was no precipitation or flocculent, etc. The MIC values of TTO nanoemulsion against Pasteurella and Salmonella were 39.65 mg/mL and 29.74 mg/mL, respectively, which were better than that of the un-nanoemulsified TTO（the MIC values for Pasteurella and Salmonella were both 49.57 mg/mL）. The results suggested that TTO nanoemulsion could solve the shortcomings of TTO such as easy volatile and difficult to dissolve in water; the prepared TTO nanoemulsion had the advantages of nano-sized particle size and uniform size, strong stability, easy storage, high dissolubility in water, and good bacteriostatic properties.