In this study, the demulsification process of four demulsifying bacteria was analyzed in situ using a newly developed method based on backscattering data from Turbiscan. First, the distribution difference of backscattering was employed to divide the entire demulsification process. Second, the Mie theory and the first order differential of the backscattering intensity along emulsion height were introduced to track emulsion variation over time. The demulsification process was divided into three stages. Consumption time, changing degree, and changing velocity during each stage varied depending on the type of bacteria. The flocculation and coalescence of water drops in the initial stage contributed substantially to the final demulsification efficiency. This study provides a powerful tool for understanding the evolution of emulsions in response to the action of various demulsifiers.