We report the development of a novel label-free electrochemical aptasensing platform to detect prostate specific antigen (PSA) based on mesoporous silica thin film-coated gold electrodes as a sensing interface. The aptasensing approach relies on hindering the diffusion of [Fe(CN)₆]^3/4− redox probe through the nanochannels of the mesoporous film gated by the recognition of the target biomarker by its anti-PSA specific DNA aptamer covalently attachment onto the outer surface of the silica nanopores. The aptasensing platform was found to be very sensitive to PSA in the range from 1 to 300 ng mL⁻¹ with a detection limit of 280 pg mL⁻¹. The aptasensor also showed high specificity, reproducibility and storage stability, and was successfully validated in spiked artificial urine samples and in blood serum from a prostate cancer-free male patient.