One kind of microbial surfactant was selected and mixed with three demulsifiers GE-189, XE-120 and XP-120, respectively, to resolved the model crude oil emulsion. The results indicated that the introduction of microbial surfactant could greatly raise the speed and efficiency of water separation from emulsion, especially at lower demulsifier concentrations, the effect of microbial surfactant on water separation from emulsion was more obvious. By means of measurement of interfacial tension, the synergetic effect between demulsifier and microbial surfactant was confirmed. Interfacial film electric capacity (Cm) and film electric resistance (Rm) were measured by electrochemical method, and the mechanism of microbial surfactant on the resolution of model crude oil emulsion was studied. The results showed that no significant effect on Cm and Rm was observed when only microbial surfactant was used, however, when microbial surfactant was mixed with demulsifier, the increase of Cm and the decrease of Rm with time were more remarkable than those only demulsifier was used, demonstrating a synergetic effect between microbial and the demulsifier. In addition, the rapid increase of Cm and the rapid decrease of Rm were closely related to the increase of water separation speed from model crude oil emulsion as a result of the addition of the microbial surfactant, which showed that the electrochemical method was feasible in studying demulsification mechanism.