Abstract We have suggested a novel method for the preparation of a label-free electrochemical immunosensor for the detection of prostate-specific antigen (PSA) as target marker for prostate cancer. Direct incorporation of PSA antibody (anti-PSA) into polypyrrole (Ppy) electropolymerized on a three-dimensional Au nanowire array has resulted in enhanced molecular interactions, ultimately leading to improved sensing performance. The electrochemical performance of the nanowire-based immunosensor array were characterized by (1) differential pulse voltammetry (DPV) to evaluate the specific recognition of PSA, (2) impedance and cyclic voltammetry to observe surface resistance and electroactivity, and (3) scanning electron microscopy (SEM) to demonstrate the three-dimensional architecture. The vertically-aligned geometric organization of Ppy provides a novel platform to improve the anti-PSA loading capacity. Overall, enhanced electrochemical performance of the proposed immunosensor has been demonstrated by its linear response over PSA concentrations ranging from 10fgmL−1 to 10ngmL−1 and a detection limit of 0.3fgmL−1, indicating that the strategy proposed here has great potential for clinical applications.