The specific mixed bacteria were domesticated from the activated sludge of coking plant using double substrate domestication. The remediation capacity of the specific bacteria on petroleum hydrocarbon contaminated soil was studied under the optimum environmental conditions. High-throughput sequencing analysis revealed that Rhodanobacter sp. was the dominant bacterium, which accounted for 41%. The optimum degradation temperature and pH were 30~35℃ and 7~8, respectively. Tween 80can be utilized as carbon source to promote the growth of the specific bacteria and improve the degradation efficiency of petroleum hydrocarbons. Moreover, Tween 80showed greater promotion on petroleum hydrocarbon degradation in contaminated soil as compared to SDS. The greatest degradation efficiency of petroleum hydrocarbons occurred at Tween 80concentration of 5CMC (i.e. 375mg/L). Furthermore, the degradation efficiency of petroleum hydrocarbons after 80days reached 77% under the optimum environmental conditions, which evidenced the outstanding remediation performance of the specific bacteria on petroleum hydrocarbon contaminated soil.