For practical applications of human embryonic stem cells (hESCs) in regenerative medicine, hESCs should be cultured on a large scale, and at the same time their properties have to be maintained in a controllable manner. Here, we report a chemically defined, scalable culture platform involving co-immobilization of heparin-catechol (HepC) and collagen type-1 (Col) for the long-term maintenance (>18 passages) of hESCs in a feeder-free condition. This platform utilizes a wet-adhesive, mussel-inspired heparin-catechol conjugate as a key component. We hypothesized that the heparin's affinity toward a wide range of proteins, might support undifferentiated in vitro growth of hESC. In fact, on the HepC-coated substrate, most hESC clumps were adhered (similar to 78% at passage 2 (P2)) and expressed pluripotency markers (Fig. 2). Although HepC alone wasn't able to support long-term maintenance of hESCs in a feeder-free system due to decrease in the adhesion rate of hESCs on HepC coating (similar to 44% at P4) during the repeated passaging processes, we found that when collagen type I was co-immobilized in the process of HepC coating, the long-term maintenance (passage 18 or more) of hESCs could be achieved with 100% adhesion efficiency (Fig. 4). One remarkable observation is that hESCs on collagen type-I underwent spontaneous differentiation after P6 (Fig. 3), which implied co-immobilized HepC played a role to suppress differentiation of hESCs. This study suggests that unlike the previous studies using proteins, peptides, or synthetic polymers, a polysaccharide, heparin, can be used as a cost-effective component for chemically defined, feeder-free culture of hESC. Statement of Significance Towards practical applications of human embryonic stem cells (hESCs) in regenerative medicine, hESCs should be cultured on a large scale, and their pluripotent property has to be maintained in a controllable manner. To address these issues, studies that develop chemically defined culture substrates have been explored to replace the widely used, complex, and undefined culture materials represented by Matrigel. Most reports have focused on utilizing proteins, peptides and/or synthetic polymers. However, there have not yet been studies on using polysaccharides as two-dimensional coating materials to potentially replace Matrigel coating. Here, we report that heparin is an effective polysaccharide for the feeder-free, two dimensional culture of hESCs. Our study implies that use of polysaccharides or a polysac-charide/ECM combination can be a new, alternative design principle for hESC culture systems.