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Abstract: In this paper, a novel, low-cost electrochemiluminescence (ECL) immunosensor using core–shell Fe3O4–Au magnetic nanoparticles (AuMNPs) as the carriers of the primary antibody of carbohydrate antigen 125 (CA125) was designed. Graphene sheet (GS) with property of good conductivity and large surface area was a captivating candidate to amplify ECL signal. We successively synthesized functionalized GS by loading large amounts of quantum dots (QDs) onto the poly (diallyldimethyl-ammonium chloride) (PDDA) coated graphene sheet (P-GS@QDs) via self-assembly electrostatic reactions, which were used to label secondary antibodies. The ECL immunosensors coupled with a microfluidic strategy exhibited a wide detection range (0.005–50UmL−1) and a low detection limit (1.2mUmL−1) with the help of an external magnetic field to gather immunosensors. The method was evaluated with clinical serum sample, receiving good correlation with results from commercially available analytical procedure. [Copyright &y& Elsevier]