The benefits of probiotics for animal health are widely recognised. In this study, we investigated the effects of dietary supplementation with Bacillus subtilis C-3102 on diet digestibility, faecal characteristics, intestinal shedding of microbiota, and hematological characteristics in healthy dogs. Adult Golden Retriever dogs (n = 18) were divided into three groups: the control group (no B. subtilis), the BS1 group (1 × 109 CFU/g of B. subtilis), and the BS2 group (2 × 109 CFU/g of B. subtilis). Dogs were fed for a 30-day administration period, and total faeces were collected in the last five days. On the first and last days of the study, blood samples were taken to determine hematological characteristics. In addition, the dry matter content, faecal consistency score, faecal output, pH, ammonia level, and quantity of short- and branched-chain fatty acids (SCFAs and BCFAs) in faecal samples were also evaluated. Results revealed that the dry matter, fibre, organic matter, and protein digestibility coefficients were higher in BS1 and BS2 (p < 0.05) compared to the control. Lower pH and higher acetate, propionate, and isobutyrate levels were detected in BS1 and BS2 (p < 0.05). Lower ammonia levels and firmer stools were observed in BS2 (p < 0.001). The numbers of faecal enterococci and lactobacilli were higher in BS1 and BS2 (p < 0.05). Furthermore, B. subtilis increased the concentrations of WBCs, RBCs, and granulocytes in BS1 and BS2 (p < 0.05). Data in the present study suggest that dietary supplementation with B. subtilis improved faecal quality, enhanced nutrient digestibility, and contributed to the dogs’ gut health by reducing gut ammonia, increasing SCFAs, and improving the number of Lactobacillus and Enterococci.HIGHLIGHTS Probiotics are one of the most commonly used feed additives in dog food. Bacillus subtilis boosts the immunity and overall gastrointestinal health of companion animals. B. subtilis dietary supplementation improved dogs’ gut health, immunity, and digestibility of food. B. subtilis increased the concentration of WBCs, RBCs, and granulocytes. B. subtilis reduced gut ammonia and increased SCFAs and the number of lactobacilli and enterococci in faeces.