Engineered surface-bound molecular gradients are of great importance for a range of biological applications. In this paper, we fabricated a polydopamine gradient on a hydrophobic surface. A microfluidic device was used to generate a covalently conjugated gradient of polydopamine (PDA), which changed the wettabilty and the surface energy of the substrate. The gradient was subsequently used to enable the spatial deposition of adhesive proteins on the surface. When seeded with human adipose mesenchymal stem cells, the PDA-graded surface induced a gradient of cell adhesion and spreading. The PDA gradient developed in this study is a promising tool for controlling cellular behavior and may be useful in various biological applications.