Abstract: Bacillus subtilis is a probiotic with important physiological functions, its activity will decrease during processing, storage and through the digestive tract. To overcome these unfavorable factors and improve the gastrointestinal survival rate and storage stability of Bacillus subtilis, this study combined nanocellulose with sodium alginate and prepared sodium alginate microcapsules (Sodium Alginate Microcapsules, S-MC) and nanocellulose sodium alginate microcapsules (Nanocellulose Sodium Alginate Microcapsules, N-MC) using endogenous emulsification method to encapsulate Bacillus subtilis. The preparation process was optimized by single factor and response surface methodology, and the two microcapsules were characterized, simulated gastrointestinal fluid test and stability were analyzed. The results showed that when the mass fraction of sodium alginate was 2.0%, the mass fraction of nano cellulose was 1.4%, the stirring speed was 610 r/min, and the volume ratio of water to oil was 1∶2.9, the embedding rate of S-MC and N-MC for Bacillus subtilis was 80.20% and 85.12%, respectively. By analyzing the results of scanning electron microscope, Fourier transform infrared spectroscopy and X-ray diffraction, it was proved that the capsule wall of S-MC had a porous structure, while nanocellulose and sodium alginate in N-MC were crosslinked with calcium ions to form a tighter gel network structure. The number of viable bacteria in N-MC decreased by 1.48 log CFU/g, which was lower than that in S-MC (2.06 log CFU/g) and free group (3.09 log CFU/g). In the simulated intestinal fluid test, S-MC released almost all Bacillus subtilis at 60 min, while N-MC released all Bacillus subtilis at 270 min, N-MC showed better slow-release performance. In addition, the storage stability test showed that N-MC could still release 7.84 log CFU/g Bacillus subtilis after 28 days of storage at 4 ℃. In this study, a new product of Bacillus subtilis microcapsule was prepared, which provided a theoretical basis for the microencapsulation of Bacillus subtilis.