Cell patterning is an important tool for biomedical research. In this work, we modified a technique combining mussel-inspired surface chemistry and microcontact printing (μCP) to modulate surface chemistry for cell patterning. Polymerized dopamine on poly(dimethylsiloxane) stamps was transferred to several cell-unfavorable substrates via μCP. Since cells only attached to the polydopamine (PDA)-imprinted areas, cell patterns were formed on a variety of cell-unfavorable surfaces. The stability of PDA imprints was proved under several harsh conditions. The cell affinity of PDA was modulated by co-deposition with several poly(ethylene imine) (PEI)-based copolymers, such as PEI, PEI-g-PEG (poly(ethylene glycol)) and PEI-g-galactose. The imprints of PDA/PEI-g-PEG provide the formation of cell patterns on cell-favorable substrates. Neuronal PC12 cells were patterned via imprinting of PDA/PEI, while HepG2/C3A cells were arranged on the imprint of PDA/PEI-g-galactose. Finally, co-culture of HepG2/C3A cells and L929 fibroblasts was accomplished by our micropatterning approach. This study demonstrated this simple and economic technique provides a powerful tool for development of functional patterned substrates for cell patterning. This technique should profit the preparation of cell patterns to study fundamental cell biology and to apply to biomedical engineering such as cell-based biosensors, diagnostic devices and tissue engineering.