The significant role of microRNAs (miRNAs) in the regulation of pathological ocular neovascularization makes them both biomarkers and therapeutic targets in vascular eye diseases. However, their acute and sensitive detection and regulation continue to be a challenge. An integrative DNA probe (detection probe) was developed to enable the single-step detection of miRNAs with high sensitivity and specificity. This probe combines the functions of specific target recognition, the DNAzyme unit-based signal reaction, and the DNA polymerase/endonuclease-assisted signal cycle. In particular, a single-stranded sequence that engages with the DNAzyme unit of the s2 sequence can identify the target miRNA and liberate the DNAzyme unit to facilitate the signal reaction with the help of metal ions. In addition, the target recycling process and signal cycle were initiated by the DNA polymerase/endonuclease-assisted chain extension and displacement process, resulting in a low limit of detection of 4.56 fM. In addition, the method demonstrated a high level of septicity for the detection of target miRNAs and was capable of distinguishing interfering miRNAs with a one-base mismatch. Our research has shown that the integrative DNAzyme nanomachine is a promising biosensor for the sensitive detection and regulation of miRNA in a single step. It is anticipated that this technology will be used in the early diagnosis and therapy of vascular eye diseases.