In order to determine the optimum growth-promoting process on lactic acid bacteria, Ramulus mori oligosaccharide was prepared by physical ultrasound, chemical acid hydrolysis and biological enzyme method, respectively. The growth-promoting effects of Ramulus mori oligosaccharide on Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus casei and Lactobacillus rhamnosus were measured. In comparison with those prepared by physical ultrasound or chemical acid hydrolysis methods, Ramulus mori oligosaccharide prepared by the biological enzyme method could specifically promote the proliferation rate of Lactobacillus rhamnosus. The Box-Behnken response surface design method and index of the proliferation rate of Lactobacillus rhamnosus were used, the optimized process for the preparation of Ramulus mori oligosaccharides by biological enzyme method was obtained as follows:β-mannanase addition reached to 50.00mg·mL-1, enzymolysis time for 4h, enzymolysis temperature at 46℃. Ramulus mori oligosaccharide was prepared under the optimized process, and the growth promoting effects of Ramulus mori oligosaccharides on Lactobacillus rhamnosus was 396.30%±0.42%, which was consistent with the predicted results, indicating that the optimized process was feasible. Studies indicated that the Ramulus mori oligosaccharide prepared by biological enzyme method could significantly increase the proliferation activity of Lactobacillus rhamnosus, and provide a certain theoretical basis for the development of Ramulus mori oligosaccharide in prebiotic food.