Formation of positively charged gold nanoparticle layers on polystyrene microparticles (PSMs600) was studied using the electrokinetic and the concentration depletion methods based on atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging. Primarily, the dependence of electrophoretic mobility of microparticles on the gold nanoparticle concentration in the suspension was measured. These results were quantitatively interpreted in terms of the three-dimensional electrokinetic model. This allowed to derive a formula for calculating the coverage of nanoparticles under in situ conditions whose validity was confirmed by direct SEM imaging of deposited gold nanoparticles (AuNPs). Additionally, the maximum coverage of gold nanoparticles for various ionic strengths was determined using a concentration depletion method based on AFM imaging of residual particles deposited on the silica substrate. The maximum coverage increased with ionic strength attaining a value of 0.35 for the ionic strength of 3 × 10-3 M. This effect was attributed to the decreasing range of lateral electrostatic interactions among deposited particles. The electrokinetic properties of the gold nanoparticle layers were also evaluated in pH cycling experiments that confirmed their stability. Beyond significance to basic science, the new data acquired in this work confirm the feasibility of preparing gold nanoparticle layers on polymer microparticles characterized by a controlled structure, coverage, and electrokinetic properties.