The influences of parameters such as the concentration of sodium alginate, the spray gas flow and the feed flow of the peristaltic pump on the size and size distribution of microcapsules were investigated. The optimal preparation parameters of microcapsules were as follows: the mass fraction was 1.5%, the spray gas flow was 450 L/h and the feed flow of the peristaltic pump was 1 mL/min. According to the optimal parameters, similar size of 30 μm and similar size distribution, which suggested that the optimal preparation parameters of microcapsules were reliable. Furthermore, the effect of different amounts of Lactobacillus acidophilus on the microcapsule's size, morphology and the encapsulation efficiency for Lactobacillus acidophilus was investigated. The results indicated that Lactobacillus acidophilus with concentration of 109 CFU/mL was optimal for encapsulation into microcapsules. After encapsulation of Lactobacillus acidophilus, the size of microcapsules was still maintained about 30 μm and the size distribution was narrow, and the encapsulation efficiency for Lactobacillus acidophilus reached 79.77%, which could be applied for gastrointestinal microenvironmental regulation.