In this study, a new disposable label-free immunosensor was developed for the electrochemical determination of alpha-fetoprotein (AFP) that is a cancer biomarker and its application was performed on human blood serum samples. In the work, firstly the screen printed carbon electrodes (SPCE) were modified with reduced graphene oxide (RGO), then the electrode surface was coated with polyneutral red (PNR) by applying the electropolymerization technique and finally, gold nanoparticles (AuNP) were created by electrodeposition method on the surface. Electrochemical characterizations of the SPCE/RGO/PNR/AuNP electrode were performed using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) methods. Immobilization of Anti-AFP to the surface of SPCE/RGO/PNR/AuNP electrodes was carried out by a covalent binding method using 3-mercaptopropionic acid (3-MPA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS). The optimum working parameters of the prepared AFP immunosensor were determined by DPV and SWV methods. It was calculated that the linear range was 1-500 ng mL-1, the limit of detections was 0.79 ng mL-1, and 0.86 ng mL1, respectively. The reproducibility of the AFP immunosensor was tested for 50 ng mL-1 AFP concentration and relative standard deviation (R.S.D.) values were calculated as 4.06% (n = 10) and 3.68% (n = 10) for DPV and SWV methods, respectively. The operation stability of the developed AFP immunosensor was examined by voltammetric methods for 60 days, and the storage stability for 12 weeks. The prepared AFP immunosensors were used for the analysis of different concentrations of AFP added to the human serum samples in a known amount and over 95% recoveries were obtained. [ABSTRACT FROM AUTHOR]