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Artificial three-dimensional (3D) tissues have the potential to be used in regenerative medicine or in vitro screening. In particular, the fabrication of 3-D cardiac tissues is greatly anticipated. However, hierarchical organization of 3-D tissues is still unknown. In regenerative medicine and drug discovery, noninvasive evaluation methods of 3-D tissues including inside of it play a key role. In this study, we report on noninvasive methods of analyzing bio-fabricated 3-D cardiac tissues using optical coherence tomography (OCT) and image analysis. Three-dimensional cardiac tissues were fabricated by coating of extracellular matrix nanofilms onto a cell surface using a layer-by-layer (LbL) technique. At first, we investigated the relationship between surface beating and its thickness to assess the value of internal analysis. The results showed that the surface beating was influenced by the thickness. Next, we tried to quantitatively evaluate the internal beating of 3-D cardiac tissues. We also confirmed the methods by changing the beating properties through the administration of isoproterenol. Our results demonstrated that the beating properties of 3-D cardiac tissues differed by depth. The results of this study suggest that information on the internal properties of 3-D cardiac tissue was necessary to understand how it functions. The combination of OCT and image analysis can be used to evaluate the internal beating properties, including changes in beating induced by a drug. It is suggested that OCT and image analysis have the potential to be used as noninvasive methods in regenerative medicine and pharmaceutical applications.