Biotin-functionalized polystyrene nanoparticles are directed by the electric field and self-assembled on the streptavidin coated microelectrode. The mean deposition percentage changes most sensitively when the level of the electrophoresis current changes. The Analysis of Variance (ANOVA) quantifies the effect of the process parameters on the assembly results. The ANOVA shows the electrophoresis current contributes most on the nanoparticle assembly by 74.42 %. Based on the ANOVA and experimental results the optimum electro-phoresis current and deposition time are determined to be 0.45 mu A and 60 s, respectively. The biotin-functionalized nanoparticles are successively deposited until the streptavidin modified electrode surface is filled. The neutravidin-functionalized nanoparticles are deposited on the biotin-functionalized nanoparticle layer in the same way. The deposition process is monitored by the fluorescent microscope. By repeating this layer-by-layer process, multilayer structure of biotin-and neutravidin-functionalized nanoparticles is able to be obtained.