The structure of tissue plays a critical role in its function and therefore a great deal of attention has been focused on engineering native tissue-like constructs for tissue engineering applications. Transfer printing of cell layers is a new technology that allows controlled transfer of cell layers cultured on smart substrates with defined shape and size onto tissue-specific defect sites. Here, the temperature-responsive swelling-deswelling of the hydrogels with groove patterns and their versatile and simple use as a template to harvest cell layers with anisotropic extracellular matrix assembly is reported. The hydrogels with a cell-interactive peptide and anisotropic groove patterns are obtained via enzymatic polymerization. The results show that the cell layer with patterns can be easily transferred to new substrates by lowering the temperature. In addition, multiple cell layers are stacked on the new substrate in a hierarchical manner and the cell layer is easily transplanted onto a subcutaneous region. These results indicate that the evaluated hydrogel can be used as a novel substrate for transfer printing of artificial tissue constructs with controlled structural integrity, which may hold potential to engineer tissue that can closely mimic native tissue architecture.