It remains as a great challenge to maintain the viability and functions of hepatocytes in vitro, and the construction of various scaffold materials and establishment of hepatocyte coculture systems are essential for the reconstruction of engineered liver devices. In our previous study, micropatterned electrospun fibrous mats were developed to control cellular behaviors, indicating that the cell growth and extracellular matrix deposition were confined to patterned regions. In the current study, the coculture of primary rat hepatocytes and fibroblasts was established on micropatterned scaffolds to modulate hepatocyte phenotype and functions. The inoculation of lactosylated poly(DL-lactide) into fiber matrices stimulated hepatocyte spheroid formation, and enhanced the albumin secretion, urea synthesis and cytochrome P-450 expression. The micropatterned coculture of hepatocytes and fibroblasts was constructed through layer-by-layer assembly of cell-loaded patterned fibers. Hepatocyte spheroids were effectively formed in the patterned regions with strong cell–cell contact, and fibroblasts were elongated in a bipolar manner and linked up into a single stretch. Compared with random coculture on fibrous mats and monoculture on patterned fibers, the coculture of hepatocytes with fibroblasts on patterned scaffolds resulted in a significantly higher amount of albumin and urea secretions and P450 expressions. Among the patterning designs, the majority of hepatocytes formed into spheroids with an average diameter of 100 μm after coculture with fibroblasts alternately located in the patterned scaffolds. This coculture system may provide a useful in vitro platform to retain the phenotype of hepatocytes and realize their functions.