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This paper proposes a high accuracy fabrication method for multilayer PDMS (polydimethylsiloxane) devices. Based on newly developed multi-stacking process using three-dimensional (3D) alignment marks of the concave-convex pair structures, a multilayer PDMS device was fabricated without skillful handling for alignment. Total misalignment of smaller than 10 μm is achieved for fabrication of a five-layer PDMS device. The precisely aligned multilayer structure was applied to a novel type of particle separation. By 3D secondary rotation flow in the 3D fluidic microchannel, two types of microparticles of 10 μm and 20 μm in diameter were successfully separated with high throughput.