Studies have been conducted to improve biosurfactant (rhamnolipid) production from Pseudomonas aeruginosa NITT 6L in liquid state fermentation utilizing palm oil and glucose as substrates. The pH and minimal salt media (MSM) compositions such as KH2PO4, K2HPO4, NaNO3, MgSO4, NaCl and FeSO4 are optimized using design of experiments. One-factor-at-a-time optimization is employed to evaluate the effects of palm oil and its co-substrate glucose on biosurfactant production. The optimal levels of the aforementioned variables are (g/L) glucose 30.0, KH2PO4 2.0, K2HPO4 5.0, NaNO3 3.5, FeSO4 0.003, MgSO4 0.2, with palm oil concentration of 1.5% (v/v). The fermentation conditions viz. period of fermentation, agitation rate, aeration and concentration of inoculum are optimized by carrying out separate experimentations. The optimum period of fermentation, agitation rate, aeration and concentration of inoculum in the fermentation medium are found to be 7 days, 150 rpm, 60% and 2% (v/v), respectively, for maximum rhamnolipid production of about 3.73 g/RE l.