Due to the simple one-step preparation method and a promising application in biomedical research, amphiphilic gradient copoly(2-oxazoline)s are gaining more and more interest compared to their analogous block copolymers. In this work, the curcumin solubilization ability was tested for a series of amphiphilic gradient copoly(2-oxazoline)s with different lengths of hydrophobic side-chains, consisting of 2-ethyl-2-oxazoline as a hydrophilic monomer and 2-(4-alkyloxyphenyl)-2-oxazoline as a hydrophobic monomer. It is shown that the length of the hydrophobic side-chain in the copolymers plays a crucial role in the loading of curcumin onto the self-assembled nanoparticles. The kinetic stability of self-assembled nanoparticles studied using FRET shows a link between their integrity and cellular uptake in human glioblastoma cells. The present study demonstrates how minor changes in the molecular structure of gradient copoly(2-oxazoline)s can lead to significant differences in the loading, stability, cytotoxicity, cellular uptake, and pharmacokinetics of nano-formulations containing curcumin. The obtained results on the behavior of the complex of gradient copoly(2-oxazoline)s and curcumin may contribute to the development of effective next-generation polymeric nanostructures for biomedical applications.