This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.