The development of a graphene-based field-effect transistor (GFET) aptasensor for prostate-specific antigen (PSA) detection. We present the novel use of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) to functionalize the graphene surface, enabling covalent bonding of amine-functionalized PSA-binding aptamers. Under optimized conditions, the shift of the GFET aptasensor’s Dirac point voltage exhibited a systematic variation with PSA concentrations. The sensor demonstrated a relatively wide linear dynamic range spanning from 100 fM to 100 nM, with a detection limit of 3.5 pM. Importantly, it displayed satisfactory specificity against potential interfering proteins and demonstrated good reproducibility and stability. Furthermore, we successfully applied the proposed method for the quantification of PSA in human serum samples, indicating its potential for bioanalysis and clinical diagnostics. The GFET aptasensor’s potential in medical diagnostics and personalized healthcare, positioning it as a promising tool for rapid and accurate biomarker detection.