The current study was designed to investigate the role of a commercial probiotic containing equal proportions (1:1) of Bacillus subtilis and B. licheniformis spores (1.6 × 10 12 CFU/kg; DiPro Aqua) on the growth, immune response, and intestinal morphology of Rainbow Trout Oncorhynchus mykiss. A total of 168 Rainbow Trout fry were randomly divided into four groups and were fed diets with different doses of DiPro Aqua (control: 0 CFU/kg; treatment 1 [T1]: 8 × 10 8 CFU/kg; T2: 16 × 10 8 CFU/kg; T3: 24 × 10 8 CFU/kg) for 56 d. The highest body weight gain and specific growth rate were obtained in the T2 and T3 groups. In addition, the highest feed conversion ratio was measured in the control group. The results showed that the serum total immunoglobulins and total protein content were increased in fish that were fed the T2 and T3 diets compared to those that received the T1 and control diets (P < 0.05). The highest alternative complement pathway hemolytic activity was measured in T2 fish compared to the other groups. According to the antibacterial activity of the skin mucus samples against Yersinia ruckeri, dietary levels of DiPro Aqua improved the mucus bactericidal activity and the maximum effect was recorded in the T2 group. The dietary levels of the probiotic mixture, especially in T2, increased the villus length, villus width, and crypt depth within the distal intestine compared to those in the control group. In conclusion, the optimal dietary dose of DiPro Aqua to boost growth performance, immunity, and intestinal morphology in Rainbow Trout was 2.0 × 10 8 CFU/kg based on polynomial regression analysis.In today's world, aquaculture plays the most crucial role in providing seafood for humans due to the growing population and the depletion of wild fishery stocks. Intensive and super-intensive aquaculture operations are developed to produce more fish and shellfish, while the most important issues faced in these production systems are poor water quality, infectious disease outbreak, and exposure to various stressors that can negatively affect the growth performance and survival rate of cultured aquatic animals (Banerjee and Ray 2017;Rico et al. 2017). In this context, fish farmers commonly use a wide range of antibiotics and chemotherapeutics for controlling and treating infectious diseases (Dawood et al. 2018;Abdel-Latif et al. 2020). In some cases, antibiotics are no longer effective in treating pathogens due to antibiotic-resistant germs (Han et al. 2015;Dawood et al. 2020). Among several alternative strategies, the use of natural feed additives such as probiotics, prebiotics, and symbiotics to improve the growth performance and immune system of fish has recently increased to make the aquaculture industry profitable and sustainable (