DNA-templated silver nanoclusters (DNA/AgNCs) serve as a useful electrochemical sensing nanomaterial characterized by excellent electroactivity and good stability, while the effect of surrounding nucleotides on their electroactivity has not been studied. Herein, we validated a nucleotide-assisted enhancement mechanism of the DNA/AgNCs electroactivity caused by T-rich nucleic acid sequences in the vicinity of AgNCs. Based on the T-rich nucleic acid-enhanced AgNCs (NAE-AgNCs) combined with hybrid chain reaction (HCR), a novel signal-enhanced electrochemical biosensing platform was established for the ultrasensitive detection of miRNA. In the presence of target miRNA-155, HCR could be triggered to generate duplex strands containing both numerous AgNC synthesis sites and T-rich overhang strands upon the electrode. With the electrodeposition of adjacent AgNCs on the electrode, the larger oxidation potential of T-rich nucleic acid leaded to stronger electron-accepting capacity, which could contribute to increased current responses. The T-rich NAE HCR electrochemical strategy resulted in a detection limit of 0.39 fM for miRNA-155 detection, one order of magnitude lower than conventional HCR-based electrochemical sensors. This T-rich nucleic acid-assisted enhancement mechanism provided a new direction to construct highly sensitive, label-free, low-cost, and simple sensing platforms for applications in biomarker assays and clinic diagnosis.