In this study, fluorinated ethylene propylene (FEP) nanoparticle as an adhesive for fabricating a threedimensional multilayered microfluidic device was studied. The formation of evenly distributed FEP nanoparticles layer with 3 μm in thickness on substrates was achieved by simple spin coating of FEP dispersion solution at 1500 rpm for 30 s. It is confirmed that FEP nanoparticles transformed into a hydrophobic thin film after thermal treatment at 300 °C for 1 hour, and fabricated polyimide film-based microfluidic device using FEP nanoparticle was endured pressure up to 2250 psi. Finally, a three-dimensional multilayered microfluidic device composed of 16 microreactors, which are difficult to fabricate with conventional photolithography, was successfully realized by simple one-step alignment of FEP coated nine polyimide films. The developed three-dimensional multilayered microfluidic device has the potential to be a powerful tool such as high-throughput screening, mass production, parallelization, and large-scale microfluidic integration for various applications in chemistry and biology.