We study the grafting of newly synthesized nickel-chelating molecules on gold surfaces via their thiol moiety. These functionalized surfaces can be used as templates to anchor recombinant proteins engineered to bear a sequence of six histidines (His-tag). To tune the nickel concentration on the surface and ultimately the protein density, we have grafted mixed monolayers in which these nickel-chelating molecules are diluted by molecules that have a similar structure but are unable to fix Ni2+ ions. We have used different complementary techniques such as contact angle measurements, ellipsometry, and high-resolution X-ray photoelectron spectroscopy (XPS) to characterize the structures of these monolayers. XPS analyses were also performed to quantify the amount of nickel fixed on the surfaces. We find that the surface composition mirrors well the relative concentrations in the grafting solution. The robustness of the complexation of the Ni2+ ions was probed by exchange and competition experiments. His-tagged protein fragments could be strongly anchored on these surfaces as probed by atomic force microscopy experiments.