HeLa cells were patterned on a microfabricated Au-coated ITO substrate. Part of the Au film was dissolved by the electrochemical wet stamping technique, and the substrate was then immersed in a thiol-terminated methoxypoly(ethylene glycol) (mPEG-SH) solution for further modification. Self-assembled monolayers (SAMs) on the gold island can form cell- and protein-resistant regions on the substrate, and living HeLa cells can adhere to the exposed ITO surface. Single cell patterns could be achieved when the size of a microstructure unit matched that of the cell. The fluorescence experiments further proved that living HeLa cells prefer to attach to the ITO substrate rather than the mPEG-SH-modified gold islands.