An electrochemiluminescent (ECL) biosensor is described for the determination of the breast cancer biomarker microRNA. The method is based on the amplification via target cyclic regeneration through a system of hairpin DNA probes, primers, and Klenow fragment of DNA polymerases combined with CdTe quantum dots (QDs) and gold nanoparticles. The assay is performed by exploiting the luminescence properties of CdTe-QDs and KSO as a co-reactive agent to increase the ECL signal. It was successfully applied to ECL-based detection of a 20-mer microRNA. The sensor has a linear response in the 0.1 fM to 0.2 pM microRNA concentration range and a detection limit as low as 33 aM. The assay has been applied to the determination of microRNA spiked in serum samples, and recoveries ranged from 94.4 to 100.5%. Graphical abstract A novel electroluminescence biosensor based on the amplification of target cyclic regeneration is described. It is achieved by using a system of hairpin DNA probes, primers, and Klenow fragment of DNA polymerases combined with CdTe QDs and Au NPs, and was successfully applied to microRNA detection.