Electrochemical aptamer-based sensors (EABs) are effective for in vitro and in vivo detection of a wide variety of targets ranging from small molecules to whole cells. Here we investigate the effect of monovalent and short chained zwitterionic molecules on the EAB sensing to differentiate structurally similar aminoglycoside and demonstrate enhanced robustness in biological media. We further integrated various blocking layers into nanoporous gold electrodes to enhance the specificity of EABs and interrogate unique signal traces arising from chemical interactions of the aptamers with various monolayers. We have found that incorporating the negatively charged monolayer (mercaptohexanoic acid (-)) can cause the aptamer to switch from its primary target to a secondary target. Using a partially positive monolayer (amino hexane thiol (+)) with a terminal amine can result in restricted aptamer binding, mostly likely through a combination of charge and hydrogen bonding effects. Incorporating zwitterionic monolayers (carboxybetaine-4/sulfobetaine-4 (±)) can result in improved antifouling. By adjusting the local electrical environment of the aptamer, we can induce changes in selectively and adjust sensor performance toward specific targets without incorporating new aptamer into the system. Additionally, we show enhanced antifouling properties upon incorporation of zwitterionic carboxybetaine-4, outperforming conventional mercaptohexanol blocking layers in whole serum while maintaining similar sensitivity toward detection of aminoglycosides. The findings of this work have will help to develop the next-generation of EABs capable of multiplex sensing of therapeutics for wearable or in situ sensing applications through modifications of the local environment with tailored blocking layers.