In this work, we report the synthesis and biophysical studies carried out on a new kind of biocompatible and very stable gold nanoparticle (GNP) stabilized with glucose through a PEG linker (AuNP-PEG-Glu). The synthetic path was optimized to obtain nanoparticles of controlled sizes. zeta-potential and dynamic light scattering measurements allowed assessment of the nanodimension, dispersity, surface charge, and stability of our GNPs. Confocal microscopy demonstrated qualitatively that glucose molecules are successfully bonded to GNP surfaces. For our study, we selected nanoparticles with diameter in a range that maximizes the internalization efficiency in cells (40 nm). A detailed investigation about the biophysical proprieties of AuNP-PEG-Glu was carried out by means of fluorescence correlation spectroscopy (FCS) and orbital tracking techniques. This work gives new insights about the uptake mechanism of gold nanoparticles capped with glucose molecules.