This study aimed to improve the heat resistance of Lactobacillus rhamnosus and its stability during the digestion process. Using the layer-by-layer (LbL) assembly technique, with zein nanoparticles (ZNP) and gum arabic (GA) as wall materials, four groups of L. rhamnosus-containing microcapsules with varying layer numbers were prepared. The microcapsules were named as (ZNP/GA)1 (one layer), (ZNP/GA)2 (two layers), (ZNP/GA)3 (three layers), and (ZNP/GA)4 (four layers). Microcapsule structure, encapsulation efficiency, and gastrointestinal tolerance were analyzed via single-factor experiments, scanning electron microscopy, and in vitro simulated digestion. The results showed that the optimal microencapsulation effect was achieved with a zein concentration of 2 mg.mL-1, a GA concentration of 2 mg.mL-1, and a pH of 4. Under these conditions, the encapsulation efficiency of the four groups of microcapsules was in the following order: (ZNP/GA)1 > (ZNP/GA)2 > (ZNP/GA)3 > (ZNP/GA)4. The number of viable bacteria was 9.53 lg colony-forming units [CFU]/mL in the (ZNP/GA)1 group, and reached 9.18 lg CFU/mL even in the (ZNP/GA)4 group. Following in vitro simulated gastrointestinal digestion for 6 h, the microcapsules with two layers of encapsulation demonstrated the best protection performance, with the number of viable bacteria being 8.34 lg CFU/mL. In conclusion, the present study demonstrated that two-layer (ZNP/GA)2 microcapsules prepared using the LbL technique provided effective encapsulation, improving the gastrointestinal tolerance of L. rhamnosus, whereas the three-layer (ZNP/GA)3 microcapsules showed better tolerance to heat treatment. The results of this study will facilitate subsequent research on protein-polysaccharide-encapsulated probiotics.