We designed a microfluidic system comprising microfluidic channels, pumps, and valves to enable the fabrication of cellular multilayers in order to reduce labor inputs for coating extracellular matrices onto adhesive cells (e.g., centrifugation). Our system was used to fabricate nanometer-sized, layer-by-layer films of the extracellular matrices on a monolayer of C2C12 myoblasts. The use of this microfluidic system allowed the fabrication of cellular multilayers in designed microfluidic channels and on commercial culture dishes. The thickness of the fabricated multilayer using this microfluidic system was higher than that of the multilayer that was formed by centrifugation. Because cellular multilayer fabrication is less laborious and the mechanical force to the cell is reduced, this novel system can be applied to tissue modeling for cell biology studies, pharmaceutical assays, and quantitative analyses of mechanical or chemical stimuli applied to multilayers.