Controlling the size of nanoscale entities is important because many properties of nanomaterials are directly related to the size of the particles. Gold nanoparticles represent classic materials and are of particular interest due to their potential application in a variety of fields. In this study, hexanethiol-capped gold nanoparticles are synthesized via the Brust–Schiffrin method. Synthesized nanoparticles were characterized by various analytical techniques such as transmission electron microscopy, scanning tunneling microscopy (STM), UV–visible absorption spectroscopy and electrochemical techniques. We have varied the molar ratio of gold to the protecting agent (hexanethiol) to discover the effect of gold-to-hexanethiol ligand ratio on the size of gold particles. The clear correlation between particle size and molar ratio is found that the averaged particle size decreases from 4.28 ± 0.83 to 1.54 ± 0.67 nm as the gold-to-ligand molar ratio changes from 1:1 to 1:9. In contrast to a recent report that thiolated gold nanoparticles are under spontaneous disintegration when they are assembled on a gold substrate, our STM experiments proved that these gold nanoparticles can form a stable monolayer or multiple layers on the platinum electrode without observing disintegration within 72 h. Therefore, our STM experiments demonstrate that the disintegration behavior of gold nanoparticles is related to the type of ligands and the nature of substrate materials. In electrochemical experiments, these gold nanoparticles displayed an electrochemical quantized charging effect, making these nanoparticles useful in the device applications such as electrochemical or biological sensors.