Three-dimensional cultures in a microfabricated environment provide in vivo-like conditions for cells, and have been used in a variety of applications in basic and clinical studies. In this study, the contractility of cardiomyocytes in a 3D environment using complex 3D hybrid biopolymer microcantilevers was quantified and compared with that observed in a 2D environment. By measuring the deflections of the microcantilevers with different surfaces and carrying out finite element modeling (FEM) of the focal pressures of the microcantilevers, it was found that the contractile force of high-density cardiomyocytes on 3D grooved surfaces was 65-85% higher than that of cardiomyocytes on flat surfaces. These results were supported by immunostaining, which showed alignment of the cytoskeleton and elongation of the nuclei, as well as by quantitative RT-PCR, which revealed that cells on the grooved surface had experienced sustained stimuli and tighter cell-to-cell interactions.