In this study, we investigate the effect of growing Aluminum nanoparticles (Al NPs) by Electron-Beam Physical Vapor Deposition (e-beam PVD) followed by thermal annealing on both Silicon (Si) and Indium Tin Oxide (ITO) substrates. Surface morphology and optical properties of the as-deposited and the thermally annealed aluminum thin-films were studied using Atomic Force Microscopy (AFM) and UV-Spectrophotometer respectively. AFM topography images of annealed Aluminum films on Si-substrate clearly show a formation of Al nanoparticles with average diameter about 40 nm while this was different in the case of Aluminum films on ITO-substrate where non-uniform flakes-like Aluminum nanostructures were observed. Other structural parameters such as surface roughness, particles distributions, and size of Aluminum nanostructures were extracted and studied for both substrates. UV-Vis absorption spectrum for annealed Aluminum film on ITO has shown a shift of about 14 nm in the absorption peak around 388 nm wavelength compared to as-deposited film. Different nanostructures in both substrates resulted from the effect of surface energy of the used substrate. Further characterization techniques will be carried out for the Al nanoparticles to provide a deep understanding of the substrate role combined with optimized properties of NPs to assist improving the control over the formation of metallic nanoparticles.