A biosurfactant-producing strain (S₆) was isolated from oil-containing wastewater in oxidation ditch and identified as Pseudomonas aeruginosa based on physiological and biochemical experiments and 16S rDNA sequence analysis. Infrared spectrum analysis revealed that S₆ produced glucolipid in the process of metabolism. It was observed that S₆ decreased the surface tension of water from 72 mN/m to 33.9 mN/m with the critical micelle concentration (CMC) of 50mg/L. The measurement of oil displacement and surface tension demonstrated that the fermented liquid had stable surface activity at varying range of salinity, pH, amount of dissolved oxygen. The optimal culture condition was obtained through orthogonal experiment: glucose 10g/L, urea 5g/L, KH₂PO₄ 1g/L, liquor of microelement 2mL, pH 8.0, water 1000mL; and the biosurfactant production under optimal culture condition was 0.173g/L.