Abstract: A crude oil-degrading bacteria ODB01 was isolated from crude oil-contaminated soil of Changqing Oilfield, capable of utilizing crude-oil as the sole source of carbon for growth, and identified as Enterobacter sp., based on physiological characteristics and the analysis of 16S rDNA gene sequence. The optimization of crude oil degradation condition was carried out by response surface methodology (Box-Behnken), the results are as follows:pH, 8.91; NaCl concentration, 1.19%; oil-bacteria liquid proportion, 1:4.12; temperature, 36.78℃, and degradation rate reaches to 34.6% under the optimum condition; In addition, adding to 0.05% Tween 80 or 0.05% Triton X-100, the degradation rates of crude oil reached to 42.5% and 46.1%, respectively. These results indicated that the bacterium ODB01 had the capability of biodegrading crude oil and it maybe developed as a remediation agent.