Modifications of biomaterial surfaces at the nanoscale offer great potential for precise control of biological events such as protein adsorption as well as cell function. A system with unique surface properties was designed here using nanosphere lithography (NSL) and layer-by-layer (LbL) technique. Tetrahedral gold nanostructures of different dimension and distance were obtained by varying the size of colloids during NSL. Polyelectrolyte multilayers (PEM) consisting poly (ethylene imine) (PEI) and heparin (HEP) were assembled on top of the structures by controlling the pH value of HEP at later stages of PEM formation, which resulted in different surface properties such as wettability and zeta potential. Further, adhesion and growth of human dermal fibroblasts was clearly affected by surface topography and chemistry. Owing to their economic effectiveness as well as simplicity, both techniques could be applied in biomedical applications to develop unique systems for stem cell therapy.