Bacillus bacteria have increasingly attracted research interest due to their promising probiotic properties, including strong antimicrobial, antidiarrheal, and immunostimulatory effects. They are able to stimulate the growth of natural flora, prevent intestinal inflammation, and demonstrate excellent stability even under adverse conditions. This chapter explores the probiotic applications of Bacillus subtilis strains in humans, animals, and plants. First, the chapter reviews the probiotic mechanisms of B. subtilis and the current state of research in this area. The potential applications of recombinant B. subtilis probiotics are also covered, along with results from preclinical studies and the future role of B. subtilis probiotic therapy in clinical settings. This chapter aims to survey the current landscape of B. subtilis probiotic research and hypothesize about its potential future impact.