This paper introduces a laser interference controlled electrochemical deposition (LIED) method for direct fabrication of periodically micro-patterned magnetite (Fe3O4) nanoparticles (NPs). In this work, Fe3O4 NPs were controllably synthesized on the areas where the photoconductive electrode was exposed to the periodically patterned interferometric laser irradiation during the electrodeposition. Thus, the micro-pattern of Fe3O4 NPs was controlled by interferometric laser pattern and the crystallization of the particles was controlled by laser interference intensity and electrochemical deposition conditions. The bottom-up electrochemical approach was combined with a top down laser interference methodology. This maskless method allows for in situ fabrication of periodically patterned magnetite NPs on the micro-scale by electrodeposition under room temperature and atmospheric pressure conditions. In the experiment, Fe3O4 NPs with the mean grain size below 100 nm in the pattern of 5 μm line array were achieved within the deposition time of 100 seconds. The experiment results have shown that the proposed method is a one-step approach in fabricating large areas of periodically micro-patterned magnetite NPs.