Biophysical and biochemical properties of the extracellular matrix (ECM) are important for regulation of cell behaviors and tissue functions. In this study, we fabricated monolayers of gelatin nanofibers on both sides of a honeycomb micro-frame by electrospinning and chemical crosslinking, resulting in tri-layer patches with adjustable thickness and pore size. Fibroblast cells were cultured on this tri-layer scaffold, showing an enhanced cell proliferation. More importantly, our results showed an efficient cell infiltration into the space between the two fiber layers and 3D cell distribution suitable for cell based assays.