Protein-polysaccharide systems show promise for probiotic encapsulation and delivery in food applications. This study investigated the formation of nanoparticles from zein and soluble soybean polysaccharide (SSPS) through antisolvent precipitation and their efficacy in encapsulating Bacillus subtilis. At pH 6.0, zein and SSPS formed composite aggregates (ZPS) that effectively encapsulated B. subtilis, as confirmed by scanning electron microscopy (SEM). Fourier-transform infrared spectroscopy (FTIR) analysis revealed the presence of hydrogen bonding and electrostatic interactions within the ZPS. The encapsulated probiotics exhibited significantly enhanced viability compared to free probiotics: 3.13-fold in simulated gastrointestinal digestion, 3.20-fold during pasteurization, and 1.50-fold in storage conditions. In vivo experiments in rats showed that oral administration of encapsulated probiotics increased the abundance of beneficial gut bacteria. These findings underscore the potential of zein-SSPS nanoparticles for probiotic protection and delivery, presenting a promising strategy for enhancing probiotic efficacy in food and nutraceutical applications.