In this paper, a novel reagentless amprometric immunosensor was built on the conductive multilayer, comprised of Nafion-coated multi-wall carbon nanotubes (MWCNT), thionine (Thi) and gold nanoparticles (nano-Au). First, Nafion-MWCNT homogeneous composite was dropped on the surface of the glass carbon electrode (GCE). Then Thi was chemisorbed by both carboxylic MWCNT and cation exchanger Nafion. Furthermore, the negative-charged nano-Au, which was used to immobilize biomolecules, was chemisorbed onto Thi film through the electrostatic force with the aminogroups of Thi. The stepwise self-assembly process of the immunosensor was characterized by means of cyclic voltammetry (CV), and the microstructure of modified film was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Using alpha-1-fetoprotein (AFP) as a model, this novel immunosensor presented amplified sensitivity, good stability, and a broader linear response in two ranges from 0.5-20 ng/mL and 20-200 ng/mL with a detection limit of 0.26 ng/mL, as well as good selectivity and storage stability. (C) 2009 Elsevier Ltd. All rights reserved.