Rhamnolipid production by Pseudomonas aeruginosa ATCC 9027 with waste frying oil as sole carbon source was studied using response surface method. Cultures were incubated in shaking flask with temperature, NO3− and Mg2+ concentrations as the variables. Meanwhile, fed-batch fermentation experiments were conducted. The results show that the three variables are closely related to rhamnolipid production. The optimal cultivation conditions are of 6.4 g/L NaNO3, 3.1 g/L MgSO4 at 32 °C, with the maximum rhamnolipid production of 6.6 g/L. The results of fed-batch fermentation experiments show that feeding the oil in two batches can enhance rhamnolipid production. The best time interval is 72 h with the maximum rhamnolipid production of 8.5 g/L. The data are potentially useful for mass production of rhamnolipid on oil waste with this bacterium.