One novel amperometric immunosensor for α-fetop- rotein was fabricated which was modified by antibody immobilized on nano ZrO2-Au-polyLysine composite membrane. Firstly, the deoxyribonucleic acid – phenoxyacetic acid isoniazid- Schiff base Co(II) complex (DNA-CoL) mixture film was casted on surface of multi carbon nanotubes(MWCNTs) modified glassy carbon electrode(GCE/CNTs/DNA-CoL). Then the composite nanoparticles (ZrO2-Au-pLL) was employed to immobilize the antibody of α-fetoprotein(AFP) to produce the probes(ZrO2-Au-pLL-anti AFP) for AFP. Finally the probes were modified on GCE/CNTs/DNA-CoL through the specific connection between DNA and ZrO2 to form a novel nanocomposite membrane immunosensor for AFP(GCE/CNTs/ DNA-CoL/ZrO2-Au–pLL-anti AFP). The function of immunosensor was investigated by scanning electron microscopy (SEM), cyclic voltammetry(CV), differential pulse voltammety (DPV) and electrochemical impedance spectroscopy experiment (EIS). The results indicated that combining the advantages of the MWNTs-DNA material and ZrO2-Au-pLL-anti AFP nano probes, the immunosensor with excellent sensitivity and selectivity to AFP was prepared successfully. The modified electrode was sensitive to AFP with a linear relationship between 0.05 and 10 ng · mL−1 and a correl- ation coefficient of 0.9905. The detection limit at a signal to noise ratio of 3 was 0.01 ng·mL−1 under the optimal conditions. The described immunosensor preparation and immunoassay methods offer promise for simple and cost-effective analysis of AFP in serum samples.