There were six high diesel oil degrading bacteria strains isolated from the oil contaminated soil that collected from Linzi City. The strain Y1 was able to produce biosurfactant rhamnolipid when cultivated on diesel oil as carbon source. The critical micelle concentrations (CMC) of rhamnolipid in water and in the soil were measured respectively according to the correlation between the surface tension of the medium and the added rhamnolipid concentration. The results showed that the CMC of rhamnolipid in water was 65 mg/L, and was 185 mg/L in soil. The tests on diesel oil biodegradation were conducted with the addition of different concentrations of rharnnolipid in water and in soil respectively. When 0.01% rhamnolipid was added to water, the diesel oil degradation was enhanced. On the contrary, when the same concentration of rhamnolipid was added to the soil, the degradation of diesel oil was inhibited. The results suggested that the rhamnolipid could enhance the diesel oil biodegradation, indicating that the concentration of rhamnolipid was higher than the corresponding CMC in the medium. Kinetics parameters for the diesel oil biodegradation parameters such as biodegradation constant (λ), coefficient of correlation (r) and half life (t1/2) in both tests were numerically analyzed in this paper, indicating that the moderate concentration of rhamnolipid in the medium could not only enhance the extent of diesel oil biodegradation but also shorten the time for oil remediation.