Graphdiyne oxide quantum dots (GDYO QDs), as derivatives of graphdiyne (GDY), have excellent electroconductibility and luminous properties and can be applied as a new ECL emitter. Herein, an electrochemiluminescence (ECL) biosensor for miRNA-21 ultrasensitive determination is constructed based on AuNPs/GDY, GDYO QD and oligonucleotide signal amplification strategy that integrates DNA walker and hybridization chain reaction (HCR) amplification. As electrode substrate material, AuNPs/GDY can not only bond with the aptamer CP but can also enhance the conductivity of the interface. When miRNA-21 exists, the DNA walker process is initiated, and the signaling probes are introduced on the electrode surface, producing abundant double-stranded H1/H2; then, H3/H4 undergoes complementary base pairing with H1/H2 through HCR. With the increase in miRNA-21, the 3D DNA nanomachine is actively manipulated, resulting in a gradual increase in ECL signal. This ECL biosensor demonstrates outstanding performance in the determination of miRNA-21 in the linear range from 0.1 fM to 1 nM. This study offers a new sensitive idea for the clinical analysis of cancer biomarkers.