High-resolution NMR has been used on the thermoresponsive hyperbranched polyethylenimines (HPEIs) modified with isobutyramide (IBAnn) groups (HPEI-IBAm), to study the structure and dynamics of the macromolecules in aqueous solution before and after the phase transition. It shows that the HPEI-IBAm macromolecule having a high IBAm substitution degree has a clear phase transition in aqueous solution, whereas the HPEI-IBAm macromolecule having a low IBAm substitution degree does not. The different phase transition behaviors have been attributed to the content as well as the distribution of the IBAm groups in the macromolecules. In order to deepen the understanding of the phase transition, the hydrophobic-hydrophobic interaction inside the HPEI-IBAm macromolecules was investigated by monitoring the H-1-H-1 NOEs between the different hydrophobic groups. An enhanced hydrophobic-hydrophobic interaction was observed in the HPEI-IBAm macromolecule having a high IBAm substitution degree after the phase transition, which provides a new perspective for our understanding of the phase transition of the macromolecules in aqueous solution. By using PFG diffusion NMR, the weight distributions of the moving particles in the solution were monitored. The beta parameter used in the PFG diffusion NMR, which reflects the change of the weight distributions of the moving particles in solution, has proved to be a good way to monitor the aggregation process of the moving particles in the solution.