The energy consumption in the biodesulfurization process was reduced by production of rhamnolipid biosurfactant in engineered biodesulfurization biocatalyst Pseudomonas aeruginosa ATCC 9027. This strain, which previously was genetically engineered for its stable biodesulfurization characteristic, was examined as having the potential to produce a type of glicolipid biosurfactant. Different environmental factors were added to examine the effects on the stability, surface tension reduction, and oil displacement of this biosurfactant. In addition, the study on the biosurfactant production time course revealed that the biosurfactant production was parallel to cell growth. However, maximum biosurfactant production occurred and was established in the stationary growth phase (after 84 h). The results demonstrated that different environmental factors, such as temperature, salinary, and aeration, barely affected the biosurfactant production and stability.