Bacillus subtilis is a bacterium with probiotic properties and health benefits, but its effectiveness can be compromised due to its susceptibility to environmental factors and stomach acid. Encapsulation safeguards the bacteria from harsh conditions and facilitates optimal therapeutic efficacy through controlled release in the gut. This study explores the development and efficacity of a nanoscale delivery system for B. subtilis probiotics nanoemulsion-based tocopherol using the binary emulsifier system (Tween 80: Gum Arabic 0.75:0.25 v/v). Key findings include a reduction of 1.9 log CFU.mL−1 for the B. subtilis nanoemulsion under simulated gastrointestinal conditions, compared to a decrease of 6.12 log CFU.mL− 1 for free cells. The encapsulated B. subtilis showcased high bacteriocin production reaching 2000 AU/mL during the stationary phase, as opposed to the average 1000 AU/mL in free B. subtilis cells. The nanoemulsion demonstrated resistance to various pH levels, retained antimicrobial activity, and exhibited high cell surface hydrophobicity along with DPPH elimination activity. The viability of encapsulated B. subtilis was significantly better under different storage conditions, maintaining a viable probiotic cell count of 7 Log CFU/mL up to 80 days. The probiotic strain showed varying susceptibility to antibiotics, indicating the potential for food safety concerns. Overall, the use of gum Arabic-based delivery system as prebiotics in the nanoemulsion formulation enhanced the performance of B. subtilis probiotics, suggesting a promising approach for the treatment of gastrointestinal disorders and for delivering viable and metabolically active probiotics to the intestine. However, further experiments are required to fully explore its potential.