Nowadays, aggregation-caused quenching (ACQ) of organic molecules in aqueous media seriously restricts their analytical and biomedical applications. In this work, hydrogen bond (H-bond) was utilized to resist the ACQ effect of 2,5,8-triamino-1,3,4,6,7,9,9b-heptaazaphenalene (Melem) as an advanced electrochemiluminescence (ECL) luminophore, whose ECL process was carefully studied in an aqueous K2S2O8 system coupled with electron paramagnetic resonance (EPR) measurements. Notably, the H-bond-induced Melem assemblies (Melem-H) showed 16.6-fold enhancement in the ECL signals as compared to the Melem aggregates (Melem-A), combined by elaborating the enhanced mechanism. On such basis, the effective ECL signal transduction was in situ achieved through the specific recognition of the double-stranded DNA embedded in Melem-H assemblies (Me-dsDNA) with spike protein (SP) of coronavirus disease 2019 (COVID-19). For that, such an ECL biosensor showed a wider linear range (1.0-125.0 pg mL-1) with a lower limit of detection (LOD) down to 0.45 pg mL-1, which also displayed acceptable results in analysis of human nasal swab samples. Therefore, the work provides a distinctive insight on addressing the ACQ effect and broadening the application scope of the organic emitter and offers a simple platform for biomedical detection.