<p>Electrochemical aptamer-based (E-AB) sensors enable the accurate measurement of target concentrations in complex matrices such as plasma or serum. However, to function effectively in these environments, the aptamers must resist nonspecific binding to proteins and other interferants. Many research literature and commercially available (“off-the-shelf”) aptamers, originally selected without consideration for biofluid compatibility, are prone to such nonspecific interactions, which diminish sensor output and compromise sensitivity. To address this challenge and expand the utility of existing aptamer sequences, we present a tunable, generalizable method to restore the EC₅₀ of E-AB sensors in plasma to levels comparable to those in buffer. We validate this approach using three therapeutically relevant small molecules: the antiretroviral emtricitabine (FTC), the antibiotic tobramycin, and the antimalarial hydroxyquinoline. Applying our method, we recovered EC₅₀ values of 11 ± 3 µM for FTC (buffer: 10.4 ± 0.6 µM), 281 ± 91 µM for tobramycin (buffer: 190 ± 16 µM), and 1.1 ± 0.6 µM for hydroxyquinoline (buffer: 900 ± 95 µM). This strategy should enable the broader application of published small-molecule-binding aptamers for biofluid measurements, regardless of their original selection conditions.</p><p></p>