Rhodococcus sp. PR-1 with high capability of demulsification on a surfactant-stabilized kerosene-water model emulsion was isolated from sewage of Dagang oil field. It could demulsify the model emulsion completely in 8 hours at 55 degrees C and had better demulsifying capability than chemical demulsifier DGF-01. The freezing-thawing and autoclaving had no effect on the process of demulsification, yet ultrasonic disposal and deal with organic solvent could inhibit its activity. A linear relationship, not the first order reaction that was used in some references, was observed between the percentage of demulsification and reaction time. The demulsifying capability of PR-1 was mainly resulted from the hydrophobic surfaces of microbial cells, which were characterized by the mycolic acids with the carbon-chain-length from 27 to 54. These results provide foundation for biological application on demulsifying crude oil emulsions in produced water of oil field.