Graphene quantum dots (GQDs) with an average diameter as small as 2.3 nm were synthesized to fabricate an electrochemiluminescence (ECL) biosensor based on T7 exonuclease-assisted cyclic amplification and three-dimensional (3D) DNA-mediated silver enhancement for microRNA (miRNA) analysis. Herein, to overcome the barrier in immobilizing GQDs, aminated 3,4,9,10-perylenetetracarboxylic acid (PTCA-NH2) was introduced to load GQDs through π-π stacking (GQDs/PTCA-NH2), realizing the solid-state GQDs application. Furthermore, Fe3O4-Au core-shell nanocomposite (Au@Fe3O4) was adopted as a probe anchor to form a novel electrochemiluminescent signal tag of GQDs/PTCA-NH2/Au@Fe3O4. The prepared ECL signal tag was decorated on the electrode surface, exhibiting excellent film-forming performance, good electronic conductivity, and favorable stability, all of which overcame the obstacle for applying GQDs in ECL biosensing and showed a satisfactory ECL response under the coreactant of S2O8(2-) (peroxydisulfate). Afterward, hairpin probe modified on the electrode was opened by helper DNA, followed by assembling target to hybridize with the exposed stem of the helper DNA. Significantly, T7 exonuclease was employed to digest the DNA/RNA duplex and trigger the target recycling without asking for a specific recognition site in the target sequence, realizing a series of RNA/DNA detections by changing the sequence of the complementary DNA. At last, the ECL signal was further enhanced by silver nanoparticles (AgNPs)-based 3D DNA networks. After the two amplifications, the ECL signal of GQDs was extraordinarily increased and the prepared biosensor achieved a high sensitivity with the detection limit of 0.83 fM. The biosensor was also explored in real samples, and the result was in good accordance with the performance of quantitative real-time polymerase chain reaction (qRT-PCR). Considering the excellent sensitivity and applicability, we believe that the proposed biosensor is a potential candidate for nucleic acid biosensing.