The biosurfactant produced by Pseudomonas aeruginosa strain KO5-2-9 could reduce surface tension to 26.6 mN/m and emulsified diesel up to 72.6% with the critical micelle concentration of 85.82 mg/L. Fourier transform infrared spectrum of extracted biosurfactant indicated the key component was glycolipid. The surface tension (ST) and emulsification index (E₂₄) were taken as evaluation indexes to study on stability of surfactants. The results showed that compared with chemical surfactants, the biosurfactant had high surface activity and emulsifying ability in high temperature, high salinity and high alkalinity. The results of physical simulation experiment indicated that the biosurfactant could enhance oil recovery over 8% and would have a good prospect in microbial enhanced oil recovery.