Surface chemical composition and roughness caused by micro/nano-topographical structures are two predominant factors in determining cellular response. A series of gold nanoparticle layers (GNPLs) with increasing surface roughness are prepared by depositing gold nanoparticles onto planar gold films. Glycine-arginine-glycine-aspartic acid-tyrosine (GRGDY) peptide is then conjugated to the surface through poly[oligo(ethylene glycol) methacrylate] polymer brushes as spacers. The pristine micro/nano-roughness structures significantly inhibit L929 cell growth. After modification with POEGMA-GRGDY, however, the micro/nano-structures greatly enhance L929 cell-specific interactions while maintaining superior low-fouling ability compared to planar gold.