Herein an ultrasensitive homogeneous ECL biosensor has been developed for TF NF-κB p50 through target-modulated proximity hybridization coupling with exonuclease III (Exo III)-powered recycling amplification. The ECL reagent (Ru(bpy)32+)-labeled hairpin DNA (HP-Rul) contains many negatively charged phosphates on the DNA chain, which cannot diffuse easily toward the negatively charged ITO electrode surface because of the large electrostatic repulsion. So a weak ECL signal can be detected. A proximity complex containing partial double strand DNA (dsDNA, as the binding site) and two hanging single-stranded DNA (ssDNA) fragments has been designed. The binding of NF-κB p50 to dsDNA effectively protects the proximity complex from digestion, forming a stable TF-DNA complex. ssDNA hybridizes with HP-Rul through proximity hybridization and hence forms a T-shape structure. This structure can be recognized by Exo III, thereby initiating the digestion process and results in the release of Ru(bpy)32+-labeled mononucleotide fragments (MFs-Rul). Meanwhile, another HP-Rul is opened and hence triggers the next cycle of hybridization and digestion process; thus, multiple MFs-Rul are generated. MFs-Rul diffuse easily to the ITO electrode because of small electrostatic repulsion, resulting in an evident signal enhancement. Under the optimal conditions, the ΔECL has a linear relationship with the logarithm of NF-κB p50 concentration ranging from 0.1 to 500 pM. The detection limit is 29 fM (S/N = 3). The sensing platform has been successfully applied to detect NF-κB p50 in cell lysates and also demonstrated to work well for NF-κB p50 inhibitor detection, exhibiting great potential in the diagnosis of disease and drug discovery.