Aptamer-functionalized field-effect transistor (FET) biosensors enable detection of small-molecule targets in complex environments such as tissue and blood. Conventional FET-based platforms suffer from Debye screening in high ionic strength physiological environments where the effective sensing distance is limited to less than a nanometer from the surface of the sensor. Aptamers that undergo significant conformational rearrangement of negatively charged backbones upon target recognition within or in close proximity to the Debye length, facilitate the transduction of electronic signals through the semiconducting channel. Herein, the fabrication of high-performance, ultrathin-film FETs and subsequent aptamer functionalization are described. Moreover, electronic sensing measurement protocols alongside calibration methods to minimize device-to-device variations are covered.