This paper introduces a laser-interference-controlled electrochemical deposition method for direct fabrication of periodically micropatterned 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 micropattern 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 microscale 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-mu m line array were achieved within the deposition time of 100 s. The experiment results have shown that the proposed method is a one-step approach in fabricating large areas of periodically micropatterned magnetite NPs.