The electrochemiluminescence(ECL) behavior of N-(4-aminobutyl)-N-ethylisoluminol(ABEI)-functionalized graphene composite(ABEI-GC) modified on an indium tin oxide(ITO) electrode was studied. ABEI-GC exhibited excellent ECL activity. On this basis, a label-free ECL immunosensor was developed for the sensitive detection of human immunoglobulin G(h Ig G) by using ABEI-GC as the ECL nano-interface via a layer-by-layer assembly technique. ABEI-GC was first assembled onto an ITO electrode. Positively charged chitosan was then electrostatically adsorbed to the modified electrode. Finally, negatively charged antibody-coated gold nanoparticles were attached to the surface to form the ECL immunosensor. In the presence of h Ig G, h Ig G was captured by its antibody. In addition, an ECL signal was detected in the presence of H2O2 when a double potential was applied. The ECL immunosensor for the determination of h Ig G showed a linear range of 1.0×10-13–1.0×10-8 g/mL with a detection limit of 5.0×10-14 g/m L. This immunosensor has high sensitivity, wide linearity and good reproducibility. The superior sensitivity of the proposed ECL immunoassay mainly derives from the incorporation of ABEI-GC, which not only improves the ECL intensity, response speed, and stability, but also provides a large specific surface for high levels of protein loading. This work reveals that ABEI-GC is good nano-interface for the construction of ECL biosensors. Our strategy is promising for protein detection and may open up a new avenue for ultrasensitive label-free immunoassays. ABEI-GC demonstrated excellent ECL activity (ABEI-GC) modified on an indium tin oxide (ITO) electrode studied. The electrochemiluminescence (ECL) behavior of N- (4-aminobutyl) -N-ethylisoluminol (ABEI) -functionalized graphene composite . On this basis, a label-free ECL immunosensor was developed for the sensitive detection of human immunoglobulin G (h Ig G) by using ABEI-GC as the ECL nano-interface via a layer-by-layer assembly technique. ABEI-GC was first charged onto an ITO electrode. Positively charged chitosan was then electrostatically adsorbed to the modified electrode. Finally, negatively charged antibody-coated gold nanoparticles were attached to the surface to form the ECL immunosensor. In the presence of h Ig G, h Ig G was captured by its antibody. In addition, an ECL signal was detected in the presence of H2O2 when a double potential was applied. The ECL immunosensor for the determination of h Ig G showed a linear range of 1.0 × 10-13-1.0 × 10-8 g / mL with a detection limit of 5. 0 × 10-14 g / m L. This immunosensor has high sensitivity, wide linearity and good reproducibility. The superior sensitivity of the proposed ECL immunoassay from the incorporation of ABEI-GC, which not only improves the ECL intensity, response speed , and stability, but also provides a large specific surface for high levels of protein loading. This work reveals that ABEI-GC is good nano-interface for the construction of ECL biosensors. Our strategy is promising for protein detection and may open up a new avenue for ultrasensitive label-free immunoassays.