Herein we demonstrate week-long operational stability of electrochemical aptamer sensors in bovine serum at 37 °C achieved by: (1) assembling the monolayers on gold with a roughness that promotes small alkylthiolate monolayer defect sizes; (2) increasing the van der Waals interaction between adjacent monolayer molecules to increase the activation energy required for desorption; (3) optimizing electrochemical measurement to mitigate both alkylthiolate oxidation and electric-field induced desorption; (4) protecting the surface from fouling by using either size-exclusion membrane protection or zwitterion antifouling chemistry. Electrochemical aptamer biosensors provide an ideal tool to investigate monolayer degradation mechanisms, as aptamer sensors require a tightly packed monolayer to preserve redox tag current versus background current, and because aptamer sensors readily reveal monolayer fouling by albumin and other solutes. As a result, this work further presents the origins and solutions for these degradation mechanisms in a logical stepwise manner that was previously unobservable over multi-day operation in biofluid.