We report a simple but straightforward approach to produce nanofiber scaffolds with incorporated protein gradient for cell culture studies. Standard electrospinning technique was used to fabricate a high-porosity random fiber matrix. Protein molecules were then deposited in the fiber matrix by a controlled filling method, allowing the generation of a concentration gradient on the sample. When cultured with NIH 3T3 cells, it was found that the cell population on the fiber matrix depends strongly on the protein concentration. The cell morphology observation also showed the effect of the hybrid system containing both a fibrillar network and surface-coated protein gradient, revealing a different behavior of cell spreading in comparison with the experimental data of cell culture without fibers or without protein gradient.