In order to solve the loss of activity of probiotics in the process of storage and feeding, as well as the competition and antagonism that may occur when two or more probiotics are mixed, and to better exert the step-by-step release and synergistic effect of two or more bacteria, Bifidobacterium adolescentis FS2-3 as the inner core microsphere and Bacillus subtilis SN15-2 as the shell microsphere were used to separate and embe the two probiotics. Sodium alginate was used as wall material and sodium mercapto carboxymethyl cellulose (CMC-SH) was added to enhance the adhesion of probiotics in intestinal tract. Probiotics double bacteria multi-core microcapsules were prepared by endogenous emulsification and extrusion. The microcapsule embedding rate was used as the evaluation index, and the optimal conditions for the preparation of probiotic double bacteria microcapsules were determined by single factor and response surface experiments. The results showed that when the concentration of sodium alginate in the inner core was 1.82%, the concentration of CMC-SH in the bacterial solution was 0.9%, the volume ratio of water to oil was 1.0∶2.9, and the concentration of sodium alginate in the outer layer was 2.9%, the embedding rate of probiotics double bacteria microcapsules could reach 72.93%. The probiotic double bacteria microcapsules under this process condition had good acid resistance and intestinal solubility. In the artificial intestinal fluid, the release rate of microcapsules reached 86.3% at 180min, which was basically completely disintegrated, and the ideal effect was achieved. The results of this study provided a reference for solving the technical problems of activity maintenance and synergism of mixed probiotic microecologics in China.