Petroleum-field emulsions, which are produced in the process of exploration, production and recovery, represent a major environmental and processing problem for the petroleum industry. Biological demulsifier as a biological agent, will become a novel method of petroleum-field emulsions demulsifier instead of chemical demulsifier for its high efficiency, low cost, high adoptability, environment-friendly, repeated use. A set of high efficiency and convenient method was established to isolate de-emulsification strain and studied on the characteristics of the de-emulsification strains. A de-emulsification strains, isolated from a petroleum-contaminated site, was evaluated for its de-emulsification capabilities using a kerosene/water model emulsion system keeping stable for more than 200 hours which was mixed by stirring Tween 60-water (0.072%, V/V) and Span 60-oil (0.028%, V/V) at the ratio (oil:water) of 6.5:3.5. We have isolated two strains with high and steady de-emulsification activity by separation and purification, blood-plate test, excluding-oil test and de-emulsifying test. The average de-emulsification rates were more than 80.0% at 24 h. The two strains are both belonging to bacillus by traditional and molecular genetic identification. By optimizing the demulsification bacteria fermentation conditions, the fermentation conditions, which is the most suitable for accumulating the fermentation products, were determined as follows: temperature 25°C, stirring speed160 r/min, pH 9, inoculated quantities 20%. When the fermentation conditions were temperature 35°C, stirring speed120 r/min, pH 9, inoculated quantities 2%, the fermentation products appeared the highest activities of demulsification.