There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass ( Yp/x), biosurfactant on sucrose ( Yp/s), and the volumetric production rate ( Y) for shake flask were obtained about 0.45 g g−1, 0.18 g g−1, and 0.03 g l−1 h−1, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Yx/s, Yp/x, Yp/s, and Y of 0.42 g g−1, 0.595 g g−1, 0.25 g g−1, and 0.057 g l−1 h−1, respectively. The biosurfactant maximum production, 2.5 g l−1, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient ( KL a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s−1, respectively. Comparison of KL a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with KL a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.