Recently, researchers have challenged to create three-dimensional (3-D) tissues with tissue engineering technology in order to establish in vitro models and new therapy for damaged organ. Most popular approach of tissue engineering is using 3-D biodegradable scaffolds as alternatives of extracellular matrix. By contrast, we have bioengineered pulsatile myocardial tissues by stacking cardiomyocyte sheets, which were harvested from temperature-responsive culture dishes only by lowering temperature. However, the shortage of oxygen and nutrition limits the final tissue thickness. In this study, we tried to fabricate thicker myocardial tissues by promoting oxygen and nutrition permeation using a novel perfusion bioreactor. Triple-layer neonatal rat cardiomyocyte sheets were attached in the culture chamber. Chambers were perfused with 2.4mL/h culture media and 15mmHg pressure gradient was applied to perfuse the culture media through the construct. After 5-days culture, the construct in the bioreactor was thicker and cell-denser than in static condition. These results indicate that the perfusion bioreactor should contribute to myocardial tissue engineering in vitro.