For potential applications in the isolation and enrichment of circulating tumor cells (CTCs), we have developed gold nanoparticle layers (GNPLs) of different roughness modified with TD05 aptamers (GNPL-APT). In serum-free binary cell mixtures containing Ramos cancer cells and CEM cells, the density of Ramos cells adherent to highly rough GNPL-APT was 19 times that of CEM cells. However, in serum-containing conditions, the specificity of GNPL-APT for Ramos cells was much reduced. To improve Ramos specificity in the presence of serum, we attached the TD05 aptamer to the layers via poly(oligo(ethylene glycol) methacrylate) (POEGMA) as an antifouling spacer (GNPL-POEGMA-APT). In serum-containing environment GNPL-POEGMA-APT showed an enhanced selectivity for Ramos cells, which increased with increasing surface roughness. The results of this study indicate that surfaces combining appropriate chemical composition and micro/nano roughness structures may be useful for cell separation, including the isolation of cancer cells for diagnosis.