Pseudomonas aeruginosa strain KO9, isolated from petroleum-contaminated soil in Karamay oil filed (China) was used to produce biosurfactant. The biosurfactant could reduce surface tension to 28 mN/m and emulsified diesel up to E24 of 81% with a critical micelle concentration of approximately 85.82 mg/L. The dried biosurfactant was light yellow powder, and the yield was 1.0866 g/L. Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the key component is glycolipid primarily consisting of lipid and carbohydrate. The crude biosurfactant showed comparable stability over a wide range of pH, temperatures and high salt concentrations, as compared to the two commercial surfactants, sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB). It has potential for use in a variety of biotechnological and industrial applications.