Pseudomonas aeruginosa ATCC9027 is an engineered strain in Iran to reduce the energy consumption in biodesulfurization, which is capable of producing the rhamnolipid biosulfactant. The biochemical analyses demonstrated that this strain reduced surface tension from 70 to 28 mN/m and emulsified hexadecane by 60%. The highest biosulfactant production was achieved when the cells were grown in minimal salt medium containing 1% (v/v) olive oil and 0.1% (w/v) yeast extract and in the presence of Isoleusin amino acid, Mg2+ (4 mg/l) and Fe2+ (0.8 g/l). Fourier transform infrared spectroscopy indicated the presence of carboxyl, amine, hydroxyl, and methoxyl groups in the extracted biosurfactant. Finally the rhlA and rhlB genes of P. aeruginosa ATCC9027 were sequenced and were highly similar to those of other strains of P. aeruginosa.