Optimization of culture conditions for the production of biosurfactant by Bacillus subtilis JK-1 was carried out using central composite rotatable design (CCRD) of response surface methodology (RSM). The variables selected for optimization of culture conditions were soluble starch, skim milk, KNO3 concentrations, and temperature. Response surface analysis showed that the fitted quadratic model had adequately high R 2 value of 0.9882 and low significance probability of <0.0001, which indicated the statistical significance of the model. The optimum levels of each variable were determined to be as follows: 1.550% (w/v) soluble starch, 0.477% (w/v) skim milk, 0.096% (w/v) KNO3 and 37.145°C. Through the statistical analysis, temperature and soluble starch were found to be the most significant factors, whereas skim milk and KNO3 had less effect within the ranges investigated. Our results could be very helpful in the large-scale production of biosurfactant from B. subtilis JK-1.