Cell sheets are useful materials in regenerative medicine; however, the cell sheet fabrication processes developed to date are associated with several crucial challenges. The aim of this study was to develop a new and simple method for the rapid and efficient fabrication of transferable monolayer cell sheets. Chemoenzymatic synthesis mediated by proteinase K was used to synthesize short cooligopeptides.for cell sheet fabrication, which showed high yield, well-defined structures, and a controllable composition. These co-oligopeptides predominantly adopted a random coil conformation in buffer. Histidine/cysteine co-oligopeptides with a disordered secondary structure displayed cysteine content-dependent esterase activity and cysteine content-independent protease activity. Taking advantage of this enzymatic activity, confluent cell monolayers were detached by simply adding the cooligopeptides solution to the culture media, and then an intact monolayer cell sheet was prepared with high cell viability and reattachment ability. The method proposed herein for preparing monolayer cell sheets represents a novel concept by using oligopeptides with enzymatic activity that show applied potential in cell sheet technology for tissue engineering and regenerative medicine.