Biosurfactants are extracellular, amphipathic surface-active agents produced by many types of microorganisms which are capable of reducing the surface and interfacial tensions. The present study aims to optimize the biosurfactant production in Pseudomonas aeruginosa DS10-129 using (2 4) factorial design. The best experimental designs were selected on the basis of response variables such as: surface tension and bacterial growth (optical density). Two types of phosphate and nitrogen sources at different concentrations were used in the experiment. Further, main effect plots and interaction plots of the selected four important factors for the study has revealed the most significant medium that influence the production of biosurfactants. It was clearly indicated that the nitrogen source is the most significant factor with greater effect on the production of rhamnolipids by P. aeruginosa among the four selected factors. Hence, it is apparent that the combination of peptone (organic nitrogen source) and high phosphate concentration with glycerol is the best for maximum production of rhamnolipid.