In the present study, poly(vinylpyrrolidone) (PVP) served as reducing and capping agents for the synthesis of gold nanoparticles (AuNPs) in alkaline medium. The PVP-AuNPs were then combined with carbon nanotubes (CNTs) to assemble thin films onto ITO via the layer-by-layer (LbL) technique. The PVP-AuNPs/CNTs LbL films were analyzed with UV-Vis spectroscopy and atomic force microscopy (AFM). The latter technique revealed that the PVP-AuNPs/CNTs LbL films took over the entire ITO surface homogeneously, which positively impacted the magnitude of the anodic currents for glycerol electro-oxidation. Such results indicated that the combination PVP-AuNPs with CNTs in an adequate LbL fashion nanostructure may be feasible for applications in various fields such as catalysis, fuel cells, and sensing.