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.