Biosurfactant producing strains were screened through enrichment from the oil sludge with crude oil as sole carbon source and 15 strains which could produce higher yield of biosurfactant were obtained. Strain BS-5 could produce highest biosurfactant and be able to reduce the surface tension of fermentation broth to 27.3 mN/m (the surface tension of pure water is 71.5 mN/m). Analytic results by FT-IR showed that the biosurfactant of the strain was a kind of glycolipid. In addition, the cultural conditions of the strain were primarily optimized by using orthogonal experiment method. The best result was: vegetable oil 10 g/L, MgSO4 0.2 g/L, K2HPO4 1.0 g/L, KH2PO4 1.0 g/L, peptone 1.0 g/L, FeSO4 0.05 g/L, CaCl2 0.02 g/L and initial pH 7.5. The strain was identified as Pseudomonas aeruginosa according to microscope observation and the 16S rDNA gene sequence.