The migration of cells is caused by the interaction of cells and the local microenvironment around them, such as changes in stiffness, chemical gradients etc. The local topography of substrates in contact with cells is a key factor that regulates the migration of cells. The interaction between the topography of the substrate and cells is crucial for the understanding of tissue development and regeneration. In this paper, the fabrication of a graded micropillar substrate for studying topography-based cell migration is described in detail. The fabrication protocol comprises of the utilization of dynamic maskless lithography system, capillary molding, and corona arc surface treatment. The fabricated micropillar substrate has been shown and the cells have been successfully seeded on the substrate. Guided cell migration on the substrate with graded microtopography has been demonstrated to occur from the sparser zone to the denser zone. Moreover, some examples of potential applications are provided.