The construction of functionalized poly(3,4-ethylenedioxythiophene) (PEDOT) nanowire devices for label-free protein detection is reported. Direct growth/assembly of PEDOT nanowires with carboxylic acid side-chain functional groups (poly(EDOT-COOH)) across the electrode junction is achieved by using an electric-field-assisted method. These functionalized PEDOT nanowire devices show typical depletion-mode p-type field-effect transistor (FET) properties. Upon conjugation with a protein-binding aptamer, the PEDOT nanowire FET devices are used for label-free electronic detection of a target protein of interest. The binding of a positively charged protein causes a substantial decrease in current flow, attributed to the specific interaction between target protein molecules and aptamer-conjugated polymer chains.