The preparation, characterization and application of a three-layer magnetic nanoparticle composed of an Fe(3)O(4) magnetic core, a Prussian Blue (PB) interlayer and a gold shell (it can be abbreviated as Au-PB-Fe(3)O(4)) for an ultrasensitive and reproducible electrochemical immunosensing fabrication were described for the first time in this work. With the employment of the Au-PB-Fe(3)O(4) nanoparticle, a new signal amplification strategy was developed based on bienzyme (horseradish peroxidase and glucose oxidase) functionalized Au-PB-Fe(3)O(4) nanoparticles for an electrochemical immunosensing fabrication by using Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model systems, respectively. The experiment results show that the multilabeled Au-PB-Fe(3)O(4) nanoparticles exhibit satisfying redox electrochemical activity and high enzyme catalysis activity, which predetermine their utility in high sensitivity antibody detection schemes. Furthermore, this immunosensor could be regenerated by simply using an external magnetic field which ensured a reproducible immunosensor with high sensitivity.