Display omitted •Easy elaboration of graphene-based aptasensor for PSA detection.•MB as a redox center for potentio-amperometric detection of PSA.•Sensitive, Selective and specific detection.•The biosensor served to detect PSA in spiked human serum. A novel and disposable potentio-amperometric aptasensor for the prostate specific antigen (PSA) was constructed using functionalized graphene-modified carbon screen-printed electrodes as transducing surface. The PSA specific DNA aptamer was covalently tethered to the graphene through amide bond between the aptamer-terminated amine and the carboxylic acid-enriched graphene casted on the electrode surface. A further hybridization of a partially complementary DNA (cDNA) was followed by intercalation of methylene blue into the double-stranded DNA sequences. The detection approach was based on a competitive assay between the antigen and the cDNA. In fact, the detection relies on the PSA biorecognition by its aptamer, triggering the release of the loosely bound DNA strand and the intercalated dye molecules, which was monitored by differential pulse voltammetry. The aptasensor allowed selective and specific detection of PSA over a wide range of concentrations from 1 pg·mL−1 to 100 ng·mL−1 with a low detection limit of 0.064 pg·mL-1. This electroanalytical device also exhibited high reproducibility and storage stability, and was successfully validated in spiked human blood serum samples.