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A disposable and magnetic nanoparticles composite membrane modified screen printed carbon electrodes (SPCEs) for rapid determination of chloramphenicol (CAP) in milk has been fabricated and characterized by scanning electron microscope (SEM) and voltammetry. The reagentless amperometric immunosensor was prepared by entrapping ferrocene (Fc) and carbon nanotubes (CNTs) into Nafion (Nf) to form a composite Nf/CNTs/Fc membrane, which yield an interface to assemble Fe3O4/Au composite nanoparticles coated chloramphenicol-bovine serum albumin (Fe3O4/Au-CAP-BSA) via an external magnetic field. Thus a reagentless amperometric immunosensor (SPCEs | Nf/CNTs/Fc | Fe3O4/Au-/CAP-BSA) for CAP was formed with competitive immunoassay. The bound Fe3O4/Au-CAP-BSA could be easily separated by an external magnetic field and used as less cost, more stable, and reusable alternatives to the soluble ones. The optimum conditions for the measurement of CAP by the immunosensor were studied. When different concentration of CAP were added in 25 muL phosphate buffer solution (PBS) containing 100 ngmiddotmL-1 CAP antibody (antiCAP), the percentage of cathodic differential pulse voltammetry (DPV) peak current increase ratio (C7%) was proportional to the concentration of CAP over the range of 0.2~80.0 ngmiddotmL-1. The detection limit was 0.11 ngmiddotmL-1. The immunosensor was employed to determine CAP in milk samples and the recovery rates exhibited a range of 88%~102%, which was consistent to the tradition HPLC method. The immunosensor was sensitive, quick, disposable with low cost, low consume of sample and easy preparation, which was suitable for screen-determination of trace CAP in food.