In this work, a novel electrochemical immunoassay system is developed for ultrasensitive detection of cancer biomarker, α-fetoprotein (AFP). This immunosensor is constructed by covalent immobilization of polyamidoamine (PAMAM) dendrimerencapsulated gold nanoparticles (Au-PAMAM) sensing interface on a Au electrode surface, followed by sequential covalent immobilization of ethyleneamineviologen (Vio) electrochemical redox marker and AFP monoclonal antibody (mAb) on the surface of Au-PAMAM via phtaloyl chloride (Pht) as linking agent. Upon the immunorecognition of the immobilized AFP to its antibody, the peak current arisen from Vio decreased due to the hindered electron transfer reaction on the electrode surface. Through the differential pulse voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments, it is found that the proposed method could detect AFP antigen with a linear range of 0.001 ng mL-1 to 45 ng mL-1 and a detection limit down to 0.3 pg mL-1. Moreover, the fabricated Au-PAMAM-based immunosensor could accurately detect AFP concentration in human serum samples demonstrated by excellent correlations with standard ELISA immunoassay. Keywords:Immunoassay,α-fetoprotein (AFP),Gold nanoparticles,Dendrimer