The demulsification of oil-in-water emulsions with microdroplets has drawn great attention as a result of their extreme stability and difficulty of separation. In this work, a series of palmitoyl-hyperbranched polyethylenimine (CHPEI) with a hyperbranched structure was developed to meet the requirement of the demulsification. The influential factors of the demulsifier concentration, sedimentation time, and temperature on demulsification were investigated to give a clear view of CHPEI-based demulsification. The results indicated that the oil removal efficiency reached 91% by adding 80 mg/L CHPEI with sedimentation time of 30 min and showed a short sedimentation time and low addition amount compared to traditional demulsifiers. Further, to better acquire the mechanism of demulsification, the oil-water interfacial tension of CHPEI demulsifiers in aqueous phase and the real-time size changes of oil droplets were both recorded. These properties make CHPEI a potential candidate as an efficient demulsifier for crude oil.