The performance and de-emulsification mechanism of alpha-amylase, a novel environmental friendly biodemulsifier in petroleum industry, was investigated at room temperature. The effects of alpha-amylase on the viscosity of amphiphilic polymer solution and de-emulsification rate were studied by changing the concentration of alpha-amylase, temperature and salinity. Polymer molecular weight, Zeta potential, interfacial film strength and interfacial tension were measured to investigate the de-emulsification mechanism of alpha-amylase. The results show that alpha-amylase is an efficient biodemulsifier to increase the de-emulsification rate of amphiphilic polymer emulsions. Hydrolysis of alpha-amylase to amphiphilic polymers destroys the structure of the amphiphilic polymer, thereby reduces the viscosity and the interfacial film strength of the system. Once de-emulsification is completed, the lower layer, i.e. the emulsified layer, will be clear. Thus, alpha-amylase can be applied as an effective de-emulsifier for amphiphilic polymer-stabilized O/W emulsion.