The fat structure of traditional infant formula is significantly different from human milk fat, which can cause the loss of fatty acids (energy) and calcium. In addition, it is not clear how ultrasonic-assisted homogenization technique affect on the properties of infant formula emulsion. To better simulate human milk fat at the triglyceride structure level, human milk fat analogue was prepared by a blend of fractionated lard and vegetable oils, and the properties of infant formula emulsion containing human milk fat analogue produced by ultrasonic-assisted homogenization technique was studied by uniform design experiments. The results showed that ultrasonic time and protein content were the main factors affecting the particle size of infant formula emulsion. The particle size decreased with increasing ultrasonic time and decreasing protein content. The minimal particle size (169.5 nm) of the emulsion was achieved under the optimum conditions determined by the regression model, which was close to the predicted value (173.8 nm). The emulsion was more stable within 7 days (the growth rate of particle size was 9%).