[en] Highlights: • The sensing strategy is based on cleavage reaction of target-assistant Zn"2"+-dependent DNAzyme. • A dual quenching mechanism of ECL is identified. • A sensitive and selective ECL sensor is constructed for detection of ATP. • The biosensor can detect ATP in serum samples with good accuracy. - Abstract: A signal-on electrochemiluminescent (ECL) approach for ultrasensitive ATP detection was developed using target-assistant Zn"2"+-dependent DNAzyme via a dual quenching pathway between quantum dots (QDs) and Au nanoclusters (Au NCs). The facile ECL biosensor was constructed by covalent assembly of Au NCs-labeled hairpin DNA on QDs modified glassy carbon electrode. A dual quenching ECL mechanism was identified to be via resonance energy transfer between QDs and Au NCs and electrocatalytic reduction of coreactant oxygen by Au NCs. With the assistance of two help DNAs, the G-quadruplex structure of ATP aptamer was formed, and thus narrowed the two fragments of Zn"2"+-dependent DNAzyme. In the presence of Zn"2"+, Zn"2"+-dependent DNAzyme can be generated in situ on the biosensor's surface. The as-prepared DNAzyme can cleave the substrate strand, and release the Au NCs from the electrode, resulting in the signal-on ECL state. This biosensor showed good analytical performance with 4 orders magnitude linear range, excellent specificity, and acceptable stability. The biosensor had been applied in detection of ATP in real serum sample and provided significant potential application in clinical analysis