Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20-33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.