Bioremediation is a low-cost, eco-friendly technique to reduce and/or eliminate pollutants from environment. Moreover, effective biological degrader for contaminants removal is important during bioremediation process. Here, the strain RHZ01, isolated from petroleum-contaminated soil in Shengli Oilfield, was identified as Rhodococcus qingshengii by the morphology, physiology, biochemistry and molecular biology. Strain RHZ01 was able to use diesel as the sole carbon source. The optimal initial pH and temperature for strain RHZ01 growth and diesel degradation were 6-8.5 and 20-30ºC, respectively. Nutrients (NaNO₃, NH₄Cl, KNO₃ and Urea) could replace NH₄NO₃. And Mg²⁺ was required for strain RHZ01 growth and diesel degradation. To investigate the diesel degrading mechanisms of this strain, the complete genome was sequenced and annotated. The complete genome consists of one chromosome with a total length 6,506,318 bp and a G+C content of 62.48%. Five putative alkane monooxygenases (AlkB) encoding genes were further analyzed, and their protein sequences were characterized and compared with other published AlkBs in Rhodococcus spp.. The physicochemical features of the five AlkBs were analyzed, and these monooxygenase proteins are alkaline, unstable and thermostable. AlkB1, AlkB2 and AlkB3 are hydrophilic protein while the other proteins were hydrophobic. All of these alkane monooxygenases share several conserved regions, such as HYG-motif and three Hist boxes. From the results, it is demonstrated that bacterial strain isolated from petroleum-contaminated could be the potential bioremediation agent for diesel removal.