The goal of this dissertation research is to investigate co-culture patterning and electrophysiological assessment methods for cardiac tissue engineering applications. Controlling and manipulating the cell microenvironment plays a fundamental role in communication, response and behavior of the in vitro cell cultures. The heart is a complex organ consists of many cell types with distinct cellular characteristics. Mimicry of the complex bio-structure of the heart in vivo requires precise manipulation techniques. In this dissertation research, we first investigate the microfabrication methods to extend the capabilities to control cell microenvironment and develop three microfabrication methods to pattern co-cultures in vivo. In the second part of this work, we extensively study the electrophysiological activity of the rat and human heart cells using both existing and original electrophysiological assessment methods. In this research, we investigate both fibroblasts and myocytes that play crucial roles in heart function. We both electrically characterize and stimulate heart cells and report our results. Finally, we design electrical circuit components utilizing cardiac cells, fabricate them using micropatterning techniques and characterized through electrophysiological assessment methods.