This study investigates the feasibility of a simple electrochemical detection of Prostate Cancer Antigen 3 (PCA3) fragments extracted from patients’ urine, using a thiolated single-strand DNA probe immobilized on a gold surface without using a redox probe. To enhance the PCA3 recognition process, we conducted a comparative analysis of the hybridization location using two thiolated DNA probes: Probe 1 targets the first 40 bases, while Probe 2 targets the fragment from bases 47 to 86. Hybridization with PCA3 followed, using square wave voltammetry. The limit of detection of the designed genosenors were of the order of (2.2 ng/mL), and (1.6 ng/mL) for Probes 1 and 2, respectively, and the subsequent sensitivities were of the order of (0.09 ± 0.01) µA⁻¹ · µg⁻¹ · mL and (0.10 ± 0.01) µA⁻¹ · µg⁻¹ · mL. Specificity tests were then conducted with the sensor functionalized with Probe 2, as it presents better analytical performances. The electrochemical results indicate that the designed sensor can clearly discriminate a complementary target from a non-complementary one. A further modeling of the calibration curves with the Power Law/Hill model indicates that the dissociation constant increases by one order of magnitude, confirming the ability of the designed sensor to perfectly discriminate complementary targets from non-complementary ones.