Myocardiocyte derived from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), is a promising cell source for cardiac tissue engineering. Combined with microfluidic technologies, a heart-on-a-chip is very likely to be developed and function as a platform for high throughput drug screening. Polydimethylsiloxane (PDMS) silicone elastomer is a widely-used biomaterial for the investigation of cell–substrate interactions and biochip fabrication. However, the intrinsic PDMS surface hydrophobicity inhibits cell adhesion on the PDMS surface, and PDMS surface modification is required for effective cell adhesion. Meanwhile, the formulation of PDMS also affects the behaviors of the cells. To fabricate PDMS-based biochips for ESC pluripotency maintenance and cardiac differentiation, PDMS surface modification and formulation were optimized in this study. We found that a polydopamine (PD) with gelatin coating greatly improved the ESC adhesion, proliferation and cardiac differentiation on its surface. In addition, different PDMS substrates varied in their surface properties, which had different impacts on ESCs, with the 40 : 1 PDMS substrate being more favorable for ESC adhesion and proliferation as well as embryoid body (EB) attachment than the other PDMS substrates. Moreover, the ESC pluripotency was best maintained on the 5 : 1 PDMS substrate, while the cardiac differentiation of the ESCs was optimal on the 40 : 1 PDMS substrate. Based on the optimized coating method and PDMS formulation, biochips with two different designs were fabricated and evaluated. Compared to the single channels, the multiple channels on the biochips could provide larger areas and accommodate more nutrients to support improved ESC pluripotency maintenance and cardiac differentiation. These results may contribute to the development of a real heart-on-a-chip for high-throughput drug screening in the future.