A reagentless amperometric immunosensor was built on a novel self-assembled sandwich nanohybrid of functionalized multi-walled carbon nanotubes（MWCNTs）,which was proposed by the following methods.Firstly,MWCNT functionalized by Nafion（N-MWCNTs） was obtained by ultrasonic method and dropped onto the surface of glass carbon electrode.After that,based on the ion exchange reaction,thionine（Thi） could be absorbed stably by both carboxylic MWCNT and cation exchanger Nafion.Following that,thionine covalently linked the nanocomposite of MWCNT functionalized by gold nanoparticles（GN-MWCNTs） through the electrostatic force with the amino groups of Thi to form the sandwich self-assembled film,which was utilized to immobilize the antibodies.After blocked by bovine serum albumin（BSA） for 30 min,the immunosensor towards for the detection of α-１-fetoprotein was successfully prepared.The α-１-fetoprotein（AFP） was immobilized on the double enlarged specific surface by GN and MWCNTs.This regentless immunosensor displayed a sensitive detection owning to the conductive electronic transformation channel constructed by the sandwich film of functionalized MWCNTs.Cyclic voltammetry（CV） was utilized to characterize the self-assemblied process of the immunosensor.And the microstructure of obtained material was observed by transmission electron microscopy（TEM）.Furthermore,the experimental conditions were optimized,including incubation time,incubation temperature and pH value of the detection solution.The proposed immunosensor presented an amplified sensitivity for the detection of AFP with a detection limit（S/N=3） of 0.12 ng?mL-1.Meanwhile,the proposed immunoassay also presented good stability,selectivity and storage stability.