The self-assembly of magnetic nanoparticles into higher-order organizations upon external magnetic stimulation has critical importance for the fabrication of discrete microstructures. In this study, the tuning of self-assembly behavior of magnetic Fe3O4 nanoparticles (MNPs), with an average size of 6 nm, under the enhanced magnetic force upon changing the applied field strength and direction is explored. Upon evaporation of the solvent where the MNPs are suspended, formation of particular micrometer sized structures is achieved with a surface constructed from sub-micrometer size magnetic beads in between the applied magnetic field and MNPs. In this study, three different surfaces fabricated using sub-micrometer size magnetic beads in between the applied magnetic field and MNPs are used and the effect of the template pattern, applied field strength and direction are explored.