Engineered tissues constructed with two-dimensionally organized cells provide promising parts for reconstructing damaged tissues. Here we propose a new method for fabricating a 2D sheet made of an endothelial cell monolayer. First a culture substrate was prepared by treating the glass surface with an amine-terminated organosilicon derivative, followed by the covalent attachment of a thin carboxymethyl cellulose (CMC) layer. Fibronectin was immobilized onto the CMC-coated surface to promote cell adhesion. These surfaces were characterized step by step by means of contact angle measurement and X-ray photoelectron spectroscopy. Porcine aortic endothelial cells adhered to the culture substrate and consequently formed a confluent monolayer. When the substrate-cell composite was immersed in a cellulase solution, a cell sheet was spontaneously detached from the substrate due to enzymatic digestion of the CMC layer. The cell-cell connections were well preserved in the cell sheet, even after detachment from the substrate, most likely due to the fact that cellulase is harmless to mammalian cells. The cell sheet could be transferred to other culture dish with the aid of a hydrophilic membrane support, retaining the proliferation activity of the cells. The results obtained in this study demonstrate that cellulase treatment of the CMC layer is a rational and efficient method for obtaining a 2D cell sheet.