Abstract: A layer-by-layer (LbL) assembly technique utilizing soy protein isolate nanofibers (SPIN) and hyaluronic acid (HA) was employed for encapsulation of the probiotic Lacticaseibacillus paracasei PC18 (PC18) to enhance its stability under processing and digestive conditions. The structure of this LbL encapsulation was characterized and the viability of PC18 during freeze-drying, exposure to acidic conditions, resistance to bile salts, and throughout the process of simulated gastrointestinal digestion were investigated. Zeta potential analysis results showed that the surface charge of PC18 fluctuated up and down with the layer-by-layer adsorption of SPIN and HA, indicating that the coating was successful. The coating was observed to be tightly adhering to the PC18 surface by transmission electron microscopy, and growth curve analysis indicated that the cells could still undergo normal metabolism after coating. The number of layers were crucial for its stability. Among, the coated PC18 with three layers, expressed as PC18(SPIN/HA)_1.5, exhibited the most superior stability. The survival rate of PC18(SPIN/HA)_1.5 after 6 h of freeze-drying was 85.04%±0.01%, significantly higher than the uncoated PC18, which was 59.07%±0.56%. After 2 h in a pH2 environment, the survival rate of PC18(SPIN/HA)_1.5 was 86.12%±0.93%, 3.34 times higher than the uncoated PC18. In 1% bile salt, the survival rate of PC18(SPIN/HA)_1.5 was 1.79 times higher than uncoated PC18 after 30 h. After a continuous in vitro gastrointestinal digestion, the survival rate of PC18(SPIN/HA)_1.5 was 56.56%±0.7%, 37.48% higher than the uncoated PC18. The stability of LbL-coated Lacticaseibacillus paracasei assemblies with SPIN and HA was significantly enhanced, particularly under freeze-drying conditions and in 1% bile salt, where PC18 encapsulated in three layers exhibited superior protective effects. This provides novel materials for the protection and delivery of probiotics, as well as provides approaches to broaden the applications of PC18.