The biological application of gold nanospheres (AuNSs) is often constrained by their stability and cytotoxicity. We present a greener synthetic approach that gives a simple and more environmentally friendly route to synthesizing AuNSs for biomedical applications. In this study, we detail a novel one-step, green synthesis of poly(2-alkyl-2-oxazoline) (POx)-coated AuNSs, which eliminates the need for additional reducing and stabilizing agents. The impact of the polymer structure on the nanoparticle formulation kinetics and nanoparticle characteristics is thoroughly investigated, revealing that the terminal functional group and the alkyl side chain significantly influence the reduction and stabilization process of AuNSs. Finally, POx-coated AuNSs were tested in vitro against F98 glioblastoma cells and proved to be usable without significant toxicity up to 75 μM. Herein, the outlined rapid and efficient method of the preparation of POx-coated AuNSs serves as a foundation for advancing the development of complex AuNSs tailored for biomedical applications.