Polymer brushes are the soft material units tethered covalently on the surface of scaffolds, which induce the functional and structural modification of a substrate's properties. Due to its facile fabrication, usability of various polymers, ECM (extracellular matrix)-like structural features, and in vivo stability, this surface coating approach has attracted special attentions in the fields of stem cell biology, tissue engineering, and regenerative medicine. Here, we summarized polymer brush-based grafting approaches comparing SAM (self-assembled monolayer)-based coating method in addition to physico-chemical characterization techniques of surfaces such as wettability, stiffness/elasticity, roughness, and chemical composition mainly affecting cell adhesion, differentiation, and proliferation. We also reviewed recent advancements of cell biological applications of polymer brushes focusing more on stem cell differentiation and 3D supports/implants for tissue formation. It is estimated that cell behaviors on polymer brushes in the scale of nanometer length contribute to the systematic understandings of simultaneous effects toward cellular responses at its interface from polymers and scaffolds for promising platform designs.