A new reagentless amperometric immunosensor for the detection of α-fetoprotein antibody （anti-AFP） molecule based on Nafion，Fe3O4@ thionine and gold nanoparticles was fabricated.Firstly，Fe3O4@ thionine nanoparticles were prepared by magnetic separation method.Nafion、Fe3O4@ thionine nanoparticles were modified on glassy carbon electrode layer by layer through electrostatic adsorption,then the NH2 of thionine molecules was used to adsorbed the gold nanoparticles.Finally，with function of gold nanoparticles adsorbing AFP antibody，a reagentless amperometric AFP immunosensor with high stability and sensitivity was prepared successfully. Transmission electron microscopy and cyclic voltammetry were used to characterize the composite nanoparticles and the property of the immunosensor.Under the optimal conditions，the immunosensor had a good current response to AFP with a detection range of 0.05-20 μg/L and a detection limit of 0.03 μg/L.