To build highly specific surfaces using aptamer affinity reagents, the effects of linker and coadsorbents were investigated for maximizing target binding and specificity for aptamer-based self-assembled monolayers (SAMs) supported on gold. An aptamer that binds the protein thrombin was utilized as a model system to compare different mixed monolayer systems toward maximizing binding and selectivity to the immobilized aptamer. Important factors used to optimize binding characteristics of thrombin to the aptamer-based monolayer films include changes in design elements of the linker and different coadsorbent thiols. Binding events measured by surface plasmon resonance (SPR) and ellipsometry showed that the binding performance of the aptamer SAMs depends principally on the linker and to a lesser extent on the coadsorbent. SAMs formed with HS-(CH2)(6)-OP(O)(2)O-(CH2CH2O)(6)-TTTTT-aptamer exhibited a 4-fold increase in binding capacity versus SAMs made using HS-(CH2)(6)-TTTTT-aptamer. Furthermore, SAMs made using HS-(CH2)(6)-OP(O)(2)O-(CH2CH2O)(6)-TTTTT-aptamer showed nearly complete specificity for thrombin versus bovine serum albumin (BSA, less than 2% bound), while a SAM incorporating a random DNA fragment (HS-(CH2)(6)-OP(O)(2)O-(CH2CH2O)(6)-TTTTT-RANDOM) showed little binding of thrombin. Irrespective of the aptamer-linker system, use of HS-(CH2)(11)(OCH2CH2)(3)OH, referred to as EG(3), as a coadsorbent enhanced binding of thrombin by approximately 2.5-fold compared to that of HS-(CH2)(6)-OH (mercaptohexanol, MCH).