A cell culture substrate which is durable and which can be readily produced in commercial scale at a low cost, and its production method are provided. The cell culture substrate comprises a substrate and a layer formed by surface modification, which comprises a polymer containing amino group produced by reacting a polymer represented by the following formula (II): (wherein n is 0 or a positive integer, and m is a positive integer, the n and m representing degree of polymerization) formed by chemical vapor deposition of formyl[2.2]paracyclophane represented by the following formula (I): (wherein k is 0 or 1) with a polymer having at least one amino group (—NH2) capable of forming Schiff base in its monomer. The production method of the cell culture substrate comprises the step of synthesizing such polymer on the substrate.