In the present study, production of rhamnolipid biosurfactant by Pseudomonas aeruginosa OG1 was statistically optimized by response surface methodology. Box-Behnken design was applied to determine the optimal concentrations of 52, 9.2, and 4.5 g/L for carbon source (waste frying oil), nitrogen source (chicken feather peptone), and KH 2 PO 4 , respectively, in production medium. Under the optimized cultivation conditions, rhamnolipid production reached up to 13.31 g/L (with an emulsification activity of 80%), which is approximately twofold higher than the yield obtained from preliminary cultivations. Hence, rhamnolipid production, noteworthy in the literature, was achieved with the use of statistical optimization on inexpensive waste materials for the first time in the present study.