S2 strain that highly produces biosurfactant was screened from the oilfield water of three plants in Daqing Oilfield in the rapid screening method, and the S2 strain was identified as Bacillus sp.. Through FT-IR and ion identification test, S2 biosurfactant was identified as nonionic lipopeptide biosurfactant. This experiment treated emulsification index (E24) of S2 strain fermentation broth as the object. Through response surface optimization method, the culture conditions (inoculum, pH, temperature and rotation speed) of the S2 strain were optimized. On the basis of response surface model, the optimum pH value was 7.2 and the temperature was 43.5 ℃, inoculation of 5.2% (V/V), rotation speed was 162 r/min through the quadratic regression equation. Under the optimal condition, the best E24 of S2 strain fermentation broth was 81.20%.