A sensitive and reusable electrochemical immunoassay for aflatoxin B(1) (AFB(1)) in food has been developed. A multifunctional magnetic bead (MMB) was initially synthesized using magnetic CoFe(2)O(4) nanoparticle as the core and Prussian blue nanoparticle (PBNP)-doped silica as the shell, and then the prepared MMB was used as an affinity support for the immobilization of the AFB(1)-bovine serum albumin conjugate (AFB(1)-BSA). With the aid of an external magnet, the AFB(1)-BSA-conjugated MMBs were attached on the surface of an indium tin oxide (ITO) electrode. Gold nanoparticles, labeled with horseradish peroxidase (HRP)-bound anti-AFB(1) antibodies (HRP-anti-AFB(1)), were employed as detection antibodies. With a competitive immunoassay format, the concentrations of AFB(1) in samples were measured in PBS (pH 7.0) by using PBNP-doped MMBs as the mediator, HRP-anti-AFB(1) as the tracer and hydrogen peroxide (H(2)O(2)) as the enzyme substrate, and the linear range was 0.05-12 ng/mL with a detection limit of 6.0 pg/mL AFB(1) (at 3sigma). Intra- and inter-assay coefficients of variation were less than 7.5%. In addition, the content of AFB(1) in red paprika specimens has been assayed by the developed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA) method, respectively, and consistent results were obtained. The as-prepared immunoassay provides a promising approach for the screening of organic pollutants because it is simple, rapid, highly sensitive, specific, and without the need of sample pre-concentration.