We report the self-assembly of a monolayer of gold nanoparticles (Au-NPs) on unmodified glass and on functionalized glass substrates with 3-aminopropyltriethoxysilane under different temperatures. The interaction between Au-NPs and APTES molecules on the glass substrates was affected by the role of APTES molecules on the glass substrates. The stability of the monolayers was carefully studied by performing a series of characterizations such as UV-Vis-NIR spectroscopy, Scanning electron microscopy (SEM). The self-assembled Au-NPs monolayers show high stability and homogenous distribution. The results of this study are important biosensing applications based on surface plasmon resonance or surface-enhanced Raman spectroscopy, where the detected signals can be adequately enhanced.