One of the probiotic bacteria is Bacillus subtilis. probiotic bacteria, when consumed, during transit through the host's digestive tract and during the stages of probiotic manufacturing and storage, the viability of this bacterium decreases which is a limitation in the use of probiotics in aquaculture. So this study was conducted to stimulate the production of indigenous B. subtilis bacteria in the digestive tract of common carp fry and its effectiveness on the survival and growth percentage of fry. Stimulation of B. subtilis production was carried out by changing the amount of nitrogen, phosphate and carbon sources, from the sources of chemical fertilizers and sugar beet molasses, each at three levels, respectively, 0.75, 1.5, 3 g/L NaNO₃ and 0.02, 0.40, 0.08 g/L K₂HPO₄ in an aqueous solution of 5% molasses. The effect of these values on the production rate of B. subtilis bacteria was investigated in laboratory conditions with the physicochemical characteristics of bile pH and simulated conditions of the digestive tract and stomach of fry. The results showed that the best combination for the production of this bacteria was with 3 grams of nitrate and 0.02 grams of phosphate in a 5% solution of beet molasses. With this prebiotic combination with 3 grams of nitrate and 0.02 grams of phosphate in a 5% solution of molasses, the highest production rate of B. subtilis bacteria was achieved. Also, by adding 1% of this prebiotic compound to the pellet food of carp fry, a statistically significant difference was observed in the growth of treated fry compared to control fry (P <0.05). Therefore, it can be concluded that the compound obtained in this study can be used as a prebiotic in bio stimulating the production of B. subtilis in the digestive tract of carp fry.