A multiplex ultrasensitive electrochemiluminescence (ECL) immunoassay was developed for the simultaneous determination of two different tumor markers, cancer antigen 153 (CA 15-3) and cancer antigen 125 (CA 125) using polyamidoamine dendrimer-quantum dots (PAMAM-QDs) and PAMAM-sulfanilic acid-Ru(bpy)3 2+ as the signal probes and Fe3O4–SiO2 as a magnetic bead. The CdTe@CdS- QDs and Ru(bpy)3 2+ at the presence of tripropyl amine (TPA) as coreactant generate ECL at an applied voltage of + 1.2 V (vs Ag/AgCl) in two different wavelengths 500 and 620 nm, respectively. Based on this strategy, the simultaneous detection of two tumor markers in single run carried out. This dual signal amplification technique was achieved by employing Fe3O4@ SiO2-dendrimer as immunosensing platform and PAMAM as the carrier for immobilizing CdTe@CdS and Ru(bpy)3 2+ probes. Experimental results illustrated that the designed immunosensor can be used to sequentially detection of CA 125 and CA 15-3 markers with the wide linear ranges of 1 μU/mL to 1 U/mL and 0.1 mU/mL to 100 U/mL with very low detection limits of 0.1 μU/mL and 10 μU/mL, respectively. The application of the immunosensor for simultaneous detection of CA125 and CA15-3 in human serum samples was evaluated and the obtained results were found to be in acceptable agreement with the those obtained with an ELISA assay as reference method. The proposed ECL immunosensor can provide a simple, sensitive and reliable approach for the simultaneous detection of tumor markers in clinical samples.