A facile method of obtaining chainlike assemblies of gold nanoparticles (AuNPs) on a chemically modified glass surface based on NaBH4 treatment is developed. Citrate-stabilized AuNPs (17 nm) are immobilized on a glutaraldehyde-functionalized glass surface and assembled into chainlike structures after treatment with aqueous sodium borohydride (NaBH4) solution. The production and morphology of the AuNP chainlike assemblies are controlled by the density of the immobilized NPs, the concentration of NaBH4 solution, and the treatment time. The AuNP assemblies are stable in water and can undergo drying. X-ray photoelectron spectroscopic data show that the number of citrate ions on the AuNPs decreased by 43% after treatment with 5 mg/mL NaBH4 solution. The NaBH4-induced partial removal of the citrate ions and the roughness of the glass surface greatly affect the binding force of AuNPs on the substrate. The immobilized AuNPs begin to move at the solid-liquid interface without desorbing when the strength of the binding force was decreased. These mobile NPs form chainlike assemblies under the driving force of van der Waals interaction and diffusion. This interface-based formation of chainlike assemblies of AuNPs may provide a simple protocol for the 1D assembly of other Au-coated colloidal nanoparticles.