Abstract: Particle size distribution of nanopowder is one of the key factors affecting microstructure and propertie of materials, and the accurate characterization of their particle sizes has been a research hotspot in this field. A measurement method for accurately characterization of the particle size and distribution of nanopowder was discussed systematically based on atomic force microscopy (AFM), with a focus on key aspects such as sample preparation, image analysis, and statistical reliability. Taking metallic nickel powder and non-metallic zirconia powder as the research objects, the problem of measurement error caused by the high surface energy of nanoparticles and their tendency to agglomerate was solved. The results showed that the uniformly distributed nanoparticles could be obtained after the dispersed liquid was dropped onto mica and dried for 30 min, after simply dispersing in water or ethanol as the solvent and ultrasonically treating for 30 min. Using the significant height difference between particles and substrate in AFM height image, each particle could be precisely separated and extracted with Image Pro software, realizing precise measurement of parameters such as the diameter and aspect ratio of individual particle. This method was applicable and highly accurate for characterizing the particle size of powder with a diameter not more than 10 nm. By analyzing the relationship between the particles number and the average particle size stability, it was concluded that the measured particle number should be not less than 1 000 in order to ensure that the average particle size results had statistical representativeness and accuracy.