Di- and triblock non-ionic copolymers based on poly(ethylene oxide) and poly(propylene oxide) were studied for the stabilization of nanoparticles in water at high ionic strength. The effect of the molecular architecture (di- vs. triblock) of these amphiphilic copolymers was investigated by using gold nanoparticles (AuNPs) as probes for colloidal stability. The results demonstrate that both di- and triblock copolymers can provide long term stability, and that in both cases AuNPs are individually embedded within globules of polymers. However, in the case of diblock copolymers, the colloidal stability was related to the formation of micelles, in contrast with the case of triblock copolymers, which were previously shown to provide good stability even at concentrations at which micelles do not form. Quartz crystal microbalance (QCM) experiments showed that the presence of the hydrophobic block in the structure of the polymer is important to ensure quantitative adsorption upon a gold surface and to limit desorption. We demonstrate that with an appropriate choice of polymer, the polymer/AuNP hybrids can also undergo filtration and freeze-drying without noticeable aggregation, which can be very convenient for further applications. Finally, preliminary studies of the cytotoxicity effect on fibroblast cells show that the polymer/AuNP hybrids were not cytotoxic. TEM micrographs on ultrathin sections of cells after incubation with the colloidal solutions show that the nanoparticles were internalized into the cells, conserving their initial size and shape.