In this research, an electrochemical PSA (prostate specific antigen) immunosensor based on Nafion/reduced graphene oxide (rGO)/aldehyde methyl pyridine (CHO-MP) composite electrode was newly developed by using noncovalent functionalization of rGO and CHO-MP. Due to improper binding stability of the immobilized antibody, immunosensors may show false positive results. The proposed composite material can provide suitable microenvironment for the immobilized anti-PSA and improve the conductivity of electrode. Furthermore, the used nafion can improve the mechanical and chemical behaviors of the developed immunosensor. Physical characterization of rGO/CHO-MP was observed through XPS, FTIR, and FE-SEM. Under the optimized conditions, the fabricated immunosensor showed a linear range of 0.005- 90 ng mL−1, detection limit of 1.6 pg mL−1, excellent selectivity, stability, and reproducibility. Lastly, human serum samples were analyzed and the recovery range was obtained as 95.8% to 107.9% with an RSD of 5.1% (n = 5). These results showed that the as-fabricated sensor is highly promising in clinical applications.