This article describes a new two-step methodology for preparing thiol monolayers having artificial recognition sites for dansylated amino acids on gold optical biosensor surfaces. N epsilon-Dansyl-L-lysine (DK) was used as the template molecule to form molecularly imprinted monolayers (MIMs). Impact factors that were studied were the concentration of DK in step one (template deposition) and the time and method for thiol monolayer formation in step two (backfilling). Compared to a prior method that used the simultaneous adsorption of the template and thiol from solution, this new approach provides the flexibility to imprint template molecules that have low binding energies on gold. Control over the surface density of imprinting sites can be achieved by this approach, and rebinding studies done using surface plasmon resonance spectroscopy confirmed that the MIMs prepared against DK showed selectivity for that template over didansyl-L-lysine.