In this work, a new electrochemical immunosensor for the detection of alpha-fetoprotein (AFP) was developed by using glassy carbon electrode modified by prussian blue-carbon nanotubes@ polydopamine (PB-CNTs@PDA). PB was used to load and coated by CNT and PDA, respectively, to increase the conductivity and specific surface area of nanocomposite, and to prevent the unstable and outflow of PB on the surface of electrode. Additionally, gold nanoparticles were synthesised on the surface of PB-CNTs@PDA dependent on excellent reduction ability of PDA coating without any other reducing agents, which provided a favourable microenvironment to increase the loading capacity of antibody without deactivation. Under optimal experimental conditions, the immunosensor shown excellent detection performance with a wide dynamic response range of AFP concentration from 0.005 to 80 ng ml −1 and a detection limit of 0.001 ng ml −1 (S/N = 3). The proposed electrochemical immunoassay method provides potential applications for other antigens or tumour markers in clinical diagnosis.