We report a sandwich-type electrochemical immunoassay for simultaneous determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), using multifunctionalized gold nanoparticles as signal tags. Two nanotags including thionine-Au nanoparticles and ferrocene-Au nanoparticles were synthesized and used as distinguishable indicators. The sensor was fabricated by means of co-immobilization of primary anti-CEA (Ab(CEA)) and anti-AFP (Ab(AFP)) antibodies on the surface of Fe3O4 nanoparticle, the strategy is based on observation distinct voltammetric peak derived from distinguish probe (Ab(CEA)-FA-Au-thionine and Ab(AFP)-Au-ferrocene nanotags). Experimental results revealed that this immunosensor enabled simultaneous determination of CEA and AFP in the linear ranges of (0.05-120 ng mL(-1)) and (0.05-100 ng(-1)), respectively. The detection limits (DL) for AFP and CEA were 0.012 ng mL(-1) and 0.018 ng mL(-1), respectively (S/N = 3). Furthermore, the methodology was also evaluated for the analysis of AFP and CEA in the clinical human serum specimens, the obtained results was in desirable agreement with clinical data.