Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes. Microorganisms synthesize them, especially during growth on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. Microbial surfactants are not yet a sustainable alternative to chemically synthesized surfactants seeing their potentially high production charges. This study highlights the use of low-cost agro-industrial raw material for fermentative production of biosurfactants. The Box–Behnken Design and response surface methodology were employed to optimize the concentrations of the ratio butter milk /distilled water, poultry-transforming wastes and inoculum size for lipopeptide biosurfactant production by B.subtilis SPB1 in submerged fermentation.The best production yield was about 12.61 ± 0.7 g/L of crude lipopeptide biosurfactant. It can be obtained when using a ratio butter milk /distilled water of 1.5, poultry-transforming wastes of 23g/L and an inoculum size of 0.12. In comparison to the highest biosurfactant production yield reported for Bacillus subtilis SPB1, three fold increases were obtained.