Electrochemiluminescence resonance energy transfer (ECL-RET) assay as an efficient analytical technique has aroused considerable interest in recognition and biosensing. In the present study, a novel self-enhanced ECL-RET of Ru(bpy)(phen-NH) as an efficient luminophore to the MoS nanosheets as effective quencher was designed for CA19-9 antigen analysis. Herein, the graphene oxide grafted hyperbranched aromatic polyamide (GO-HBP) with high loading ability for Ru-complex was used as the sensing platform, while amine-rich nitrogen-doped carbon nanodots (NCNDs) which covalently linked to Ru(bpy)(phen-NH) applied as co-reactant for enhancing of anodic ECL signal response. In this approach GO-HBP-Ru-complex-NCND-anti-CA19-9 Ab exhibited amplified ECL emissions ("on" state) and with formation of sandwiched immunocomplex between, immobilized CA19-9 Ab, CA19-9 antigen and MoS nanosheets modified with CA19 9 antibody, the ECL response of luminophore was efficiently quenched ("off" state). The signal response was doubly amplified by covalent attachment of more luminophore, co-reactant and CA19-9 antibody with hyperbranched aromatic polyamide. On the basis of all above features, the ECL intensity of Ru-NCND decreased with the increase of the concentration of CA19-9 antigen in a wide linear range of 2 mU mL- 50 U mL with the detection limit of 0.25 mU mL (S/N = 3). The application of the fabricated ECL-RET immunosensor for determination of CA19-9 antigen in human serum samples was appraised that the satisfactory results were found to be in admissible accord with those gained with the reference method (ELISA assay).The suggested method opens up a novel avenue for expanding high-performance ECL-RET immunosensors cancer markers detection in clinical monitoring.