One demulsifying strain of Alcaligenes sp. S-XJ-1, isolated from the oil-contaminated soil, could grow at initial cultivation pH ranging from 6.0 to 11.0 and the optimum pH was observed at 10.0. Cultivated at initial pH 10.0, the biomass yield reached highest at 4.8 g/L and the emulsion breaking ratio achieved over 85% within 24 h when the biodemulsifier was dosed at 1000 mg/L. Meanwhile, this demulsifying strain S-XJ-1 had a higher cell surface hydrophobicity with ratio of microbial adhesion to hydrocarbon at 72.7% and water contact angle at 115° when it was cultivated at initial pH 10.0. To compare the demulsification process of demulsifiers produced at pH 7.0 and pH 10.0, kerosene-water model emulsion (W/O) mixed with the demulsifier was analyzed using Turbiscan method. Compared with the biodemulsifier produced under initial pH 7.0, the biodemulsifier produced at initial pH 10.0 remarkably increased the particle size of the dispersed phase, and then greatly improved the emulsion breaking rate.