In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to control living adherent cells on a culture substrate by irradiating light onto multiple points simultaneously. Among them, we developed a functional cell culture surface, on which polyethylene glycol as a cell adhesion inhibitor is immobilized via a photo-responsive polymer. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-adhesive area can be appended even after cell seeding just by irradiating light. Additionally to the simple cell patterning, construction of patterned co-culture system was demonstrated by using the substrate.