The study objective is studied the probiotic characteristics of Bacillus subtilis BSO2 isolated from human faeces. Probiotic characteristics were tested according to the guidelines of WHO/FAO, which include: producing extracellular enzymes, competitive ability against pathogenic bacteria in gastrointestinal tract, tolerance with acidity in the stomach and bile salt, and safety aspects. After that, the ability to treat diarrhea in a mouse model of BSO2 spores was investigated. The results showed that BSO2 can produce extracellular enzymes, survive in the strict conditions of gastrointestinal tract. The antibiotic resistance test results showed that BSO2 sensitive to 13 tested antibiotics, except ceftazidime. Ultimately, BSO2 spores had capable of treating diarrhea in mouse model when compared with control group not used probiotic.