Transplantation of cell sheets including an intact extracellular matrix is one tissue-engineering strategy for tissue regeneration. Temperature-responsive substrates based on poly(N-isopropylacrylamide) (PNIPAAm) have been used to harvest intact cell sheets by temperature change. In this work, we immobilized PNIPAAm on plastic substrates by a UV-activated azide-based cross-linking mechanism. We demonstrated that the UV-cross-linked PNIPAAm films could respond to temperature changes and be used for cell-sheet fabrication. Next, grooved PNIPAAm substrates were fabricated by imprinting from grooved poly(dimethylsiloxane) (PDMS) molds (800 nm in groove width and 500 nm in depth). C2C12 cells formed aligned cell sheets on the grooved PNIPAAm surface. The aligned cell sheet could be transferred to a gelatin substrate without losing cell alignment. We expect that this simple time-saving technique for the fabrication of grooved PNIPAAm substrates will benefit from the application of cellular alignment in tissue-engineering products.