A graphene oxide-Ru(bpy)32+-polyaniline-nanocomposite (GO-Ru(bpy)32+-PANI) was prepared to develop a sensitive label-free electrochemiluminescence (ECL) immunosensor for mucoprotein 1 (MUC1). Ru(bpy)32+ and PANI were decorated together on graphene oxide for the first time. The GO-Ru(bpy)32+-PANI material was characterized by scanning electron microscopy (SEM), absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), fluorescence, electrochemical impedance spectroscopy (EIS), and ECL. The GO-Ru(bpy)32+-PANI exhibited excellent stability. The ECL immunosensor for MUC1 was fabricated by covalently immobilizing anti-MUC1 upon GO-Ru(bpy)32+-PANI modified indium-doped tin oxide (ITO) electrode. In the presence of MUC1, a decrease of ECL was observed due to the formation of the anti-MUC1/MUC1 complex. The immunosensor showed a linear relationship from 1 × 10−13 to 1 × 10−7 mg/mL with a detection limit of 8.2 × 10−14 mg/mL. The immunosensor was applied to the determination of MUC1 in human serum. [ABSTRACT FROM AUTHOR]