Prostate cancer is one of the most frequently diagnosed male malignancies and can be detected by prostate-specific antigen (PSA) as a biomarker. To detect PSA, several studies have proposed using antibodies, which are not economical and require a long reaction time. In this study, we propose to use self-assembled thiolated single-strand DNA on electrodes functionalized by multi-walled carbon nanotubes (MWCNT) modified with gold nanoparticles (AuNPs) to realize a low-cost label-free electrochemical biosensor. In this regard, the PSA aptamer was immobilized via electrostatic adsorption on the surface of a screen-printed MWCNT/AuNPs electrode. The immobilization process was enhanced due to the presence of Au nanoparticles on the surface of the electrode. Surface characterization of the electrode at different stages of modification was performed by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) and contact angle for surface tension properties. The results showed an increase in surface roughness due to the absorbance of the aptamer on the electrode surfaces. The developed sensor has an extended linear range of 1-100 ng/mL, and a very low limit of detection down to 1 pg/mL. In addition, the reaction has a binding time of only five minutes on the developed electrodes. Investigations of the biosensor selectivity against several substances revealed an efficient selectivity for PSA detection. With this approach, low-cost biosensors with high sensitivity can be realized which have a wide linearity range and a low limit of detection, which are necessary for the early detection of prostate cancer.