MiRNAs play crucial roles in cell proliferation, metabolism, and signal transduction, and have been established as biomarkers for cancer diagnosis and treatment for many years. Traditional methods for detecting miRNAs have several drawbacks, including poor sensitivity, time-consuming processes, and laborious steps. We have combined nicking enzyme amplifica-tion reaction (NEAR) with DNAzyme to develop a single-tube detection platform for highly sensitive and rapid detection of miRNA-21. The target miRNA activates NEAR amplification to produce many single-stranded DNAs, which hybridize to another DNA strand in the system to form a cleavage-active magnesium ion-dependent DNAzyme. When magnesium ions are present in the system, DNAzyme degrades the FQ reporter to generate a fluorescent signal to achieve highly sensitive and rapid detection of miRNA. This assay enables highly sensitive detection of miRNAs with a detection limit of 1.91 pM. The assay is one-tube reaction, and miRNA detection can be completed in approximately 30 minutes at room temperature. These results indicate that this biosensor has a broad application prospect in cancer diagnosis and treatment based on miRNA detection.