Abstract: Three strain diesel degrading bacteria could be isolated by a series of conventional screening step including enrichment culture, morphologic observation, hydrolyzing oil spot measurement, diesel oil degradation rate assay etc. Then the fermentation supernatant of first-isolated strains was secondly screened by several screening methods including drop-collapsing method, surface tension method, oil displacement activity and emulsification index. One strain diesel degrading bacteria was isolated with higher surface activity from oil-contaminated seawater and sediments in the Zhoushan fishery, Zhejiang Province, China. This isolated strain was identified as Yarrowia lipolytica based on the analysis of its 18s rRNA sequence. This strain showed strong hydrolyzing oil activity, and the degradation rate of the diesel oil was 80%. The surface tension could be reduced from 73.4 mN/m to 23.56 mN/m, and E₂₄ reach 60%. The results of TLC and IR analysis showed that biosurfactant of the isolated strain was lipopeptide and the results of GC/MS showed that the surfactant was composed of C₁₄、C₁₅β hydroxy fatty acid.