With a two-phase method, well-dispersed 5.2 nm Ag nanoparticles with narrow size distribution (±0.5 nm) are synthesized. The assembled structures of colloidal Ag nanoparticles on highly oriented pyrolytic graphite (HOPG), silicon chip and microscopic glass have been investigated by atomic force microscopy (AFM). With different spin-coating speeds and concentrations of colloidal silver nanoparticles, various assembly structures could form on those substrates. On HOPG, Ag nanoparticles were absorbed and aligned along single-atom-height step edges to form a linear one-layer structure. And on silicon chip and microscopic glass, one-layer closed packing fractal structure and two-layer closed packing ring were observed respectively.