Four bacterial strains (X1, X2, X3 and X4)using diesel oil as sole carbon source were screened from soil, which was polluted by oily wastewater, and they were identified as Serratia sp., Acinetobacter sp., Bacillus sp. and Azomonas sp., respectively. After strain X4 was cultured for 28 d with a rotary shaker at 32°C, the degradation rate of diesel oil by the strain reached 62% and also the biodegradation of diesel oil was significantly enhanced by the amendment of rhamnolipid biosurfactant, which indicated that rhamnolipid increased microbial biomass and the biodegradation of diesel oil was 26% higher than that without rhamnolipid. Adding rhamnolipid could shorten the half life by nearly 50%. By designing orthogonal tests, the effects of four major factors, culture temperature, culture time, dosage of rhamnolipid and concentration of petroleum hydrocarbons, on the degradation rate of hydrocarbons in water were studied.