The relationship between demulsification performance and dynamic interfacial activity of polyether demulsifiers is essential. Herein, a series of hyperbranched poly(amido amine) polyethers (PAM polyethers) were designed and synthesized by controlling the mass ratio of propylene oxide (PO) to ethylene oxide (EO). The results indicate that the dehydration efficiency increased gradually with increasing the EO content, and PAM9915 can achieve complete separation of crude oil and water at 60 min. Therefore, the optimal polyether demulsifiers have a specific relative solubility number (RSN) value (≈ 18.5) to attain hydrophilic-lipophilic deviation (HLD) = 0, which other types of polyethers confirmed. On the other hand, when the PO and EO mass ratio equals 1.5, the demulsifier has an overdosing effect with a concentration above 50 mg·L−1. In addition, the dynamic interfacial tension (DIT) results show that dehydration efficiency has a positive correlation with dynamic interfacial activity. This work provides deep insight into physicochemical mechanisms needed to obtain effective demulsification and could help guide the workload of the demulsifier screening.