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This work describes a sensitive voltammetric immunosensor for label‐free electroanalysis of the prostate specific antigen (PSA), the main biomarker of prostate cancer. A gold electrode was firstly modified with the optimum self‐assembled monolayer (SAM), 1,6‐hexanedithiol, followed by the subsequent adsorption of gold nanoparticles (AuNPs) and, then, the monoclonal antibody to recognize PSA was immobilized. The influence of the most significant experimental variables (SAM type and incubation time, AuNPs deposition, antibody concentration and bovine serum albumin immobilization) on the biosensor response was studied by microscopy and voltammetry techniques. The electroanalytical detection was based on the interaction between PSA antibody and PSA via square‐wave voltammetry using ferrocyanide/ferricyanide as electrochemical redox indicator. Using the proposed immunosensor, PSA was specifically detected within the linear range between 0.2 and 200 ng mL−1 with 0.01 ng mL−1 as limit of detection. The immunosensor allows accurate, reproducible and sensitive (22.7 % reduction mL ng−1) detection in a concentration range useful for clinical purposes. [ABSTRACT FROM AUTHOR]