Interest in biosurfactants has increased considerably in recent years, as they are potentially used in many commercial applications in petroleum, pharmaceuticals, biomedical, and food processing industries. Since improvement of their production was of great importance to reduce the final coast, cultural conditions were analyzed to optimize biosurfactants production from Bacillus subtilis SPB1 strain. A high yield of biosurfactants was obtained from a culture of B. subtilis using carbohydrate substrate as a carbon source; among carbohydrates, glucose enhanced the best surfactin production. The optimum glucose concentration was 40 g/L. Higher amount of biosurfactants was obtained using 5 g/L of urea as organic nitrogen source and applying C/N ratio of 7 with ammonium chloride as inorganic nitrogen source. The highest amount of biosurfactants was recorded with the addition of 2% kerosene. Moreover, it was shown, using an automated full-controlled 2.6 L fermenter, that aeration of the medium, which affected strongly the growth regulated biosurfactants synthesis by the producing cell. So that, low or high aerations lead to a decrease of biosurfactants synthesis yields. It was found that when using dissolved oxygen saturation of the medium at 30%, biosurfactants production reached 4.92 g/L.