Two kinds of hyperbranched poly(amido amine) (h-PAMAM) with abundant terminal amine groups are synthesized to break the oil-in-water (O/W) emulsions consisting of microdroplets. The demulsification performance of h-PAMAM is systematically evaluated with assistance of UV-vis spectra, micrograph, DLS and SAXS analyses. The demulsifier with 1,3-propanediamine as initiator shows a striking demulsification ability with small dosage (<40 mg/L). The demulsification equilibrium can be obtained within 30 min, and oil removal ratio reaches to 92%, which is ascribed to the highly hyperbranched structure and the adequate molecular weight. The demulsifier molecules with hyperbranched structure easily diffuse to the oil-water interface and reveal a high interfacial activity, which causes film break and coalescence of oil droplets, and consequently leads to high oil removal ratio. The combination of UV-vis spectra, micrograph, DLS and SAXS analyses provides a new approach to monitor demulsification process and comprehend demulsification mechanism. The excellent demulsification performance of h-PAMAM shows a considerable potential applications of this hyperbranched polymer for wastewater treatment and petroleum industry.