A novel electrochemical immunosensor for tumor biomarker detection based on three-dimensional, magnetic and electroactive nanoprobes was developed in this study. To fabricate the nanoprobes, negatively charged Fe3O4 nanoparticles (Fe3O4 NPs) and gold nanoparticles (Au NPs) were first loaded on the surface of multiple wall carbon nanotubes (MCNTs) which were functioned with redox-active hemin and cationic polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA). Using alpha fetoprotein (AFP) as a model analyte, AFP antibody (anti-AFP) was absorbed on the surface of Au NPs, bovine serum albumin (BSA) was then used to block sites against non-specific binding, and finally formed anti-AFP/Au NPs/Fe3O4/hemin/MCNTs named anti-AFP nanoprobes. When the target antigen AFP was present, it interacted with anti-AFP and formed an antigen-antibody complex on the nanoprobe interface. This resulted in a decreased electrochemical signal of hemin for quantitative determination of AFP when immobilized onto the screen-printed working electrode (SPCE). The results showed that the nanoprobe-based electrochemical immunosensor was sensitive to AFP detection at a concentration of 0.1 to 200 ng.mL(-1) with a detection limit of 0.04 ng.mL(-1), it also demonstrated good selectivity against other interferential substances. The electroactive nanoprobes can be massively