Provided is a temperature-responsive cell culture substrate. A non-crosslinked temperature-responsive polymer having a molecular weight between 10,000 and 150,000 is immobilized on the substrate surface with a density of 0.02 to 0.3 molecular chain per square nanometer. Using the provided temperature-responsive cell culture substrate, cells obtained from various tissues can be efficiently cultured. This culturing method makes it possible to efficiently peel off a cell sheet by just changing the temperature, without causing damage.