Abstract: Scanning Electron Microscopy (SEM) is a key tool for characterizing the microstructure, size distribution, and surface defects of polystyrene (PS) microspheres. However, the poor conductivity of PS microspheres easily leads to charging effects under high-energy electron beams, causing image distortion, bright spots, and other issues. Although traditional coating can improve conductivity, it obscures surface details. This study systematically investigated the wet sample preparation and optimization of SEM testing parameters for monodisperse micron-sized PS microspheres synthesized by dispersion polymerization, aiming to obtain high-quality images while avoiding coating. The study shows that the dispersion state of PS microspheres is significantly influenced by concentration, particle size, and solvent evaporation rate. Low-concentration samples treated with ultrasound can achieve a uniform monolayer distribution of PS microspheres. Regarding SEM testing, exploring the impact of key parameters (such as accelerating voltage, working distance, and detector selection) on imaging uncoated samples revealed that using a Zeiss Sigma 300 SEM with a low accelerating voltage (1 kV), combined with the SE2 detector and a working distance of (5～7) mm, yields high-resolution images free from severe distortion without the need for surface gold sputtering. This enhances the efficiency and accuracy of PS microsphere characterization.