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The abnormal expression of miRNA is closely related to the occurrence of pancreatic cancer. Herein, a programmable DNAzyme amplifier for the universal detection of pancreatic cancer-related miRNAs was proposed based on its programmability through the rational design of sequences. The fluorescence signal recovery of the DNAzyme amplifier showed a good linear relationship with the concentration of miR-10b in the range of 10–60 nM, with a detection limit of 893 pM. At the same time, this method displayed a high selectivity for miR-10b, with a remarkable discrimination of a single nucleotide difference. Furthermore, this method was also successfully used to detect miR-21 in the range of 10–60 nM based on the programmability of the DNA amplifier, exhibiting the universal application feasibility of this design. Overall, the proposed programmable DNAzyme cycle amplifier strategy shows promising potential for the simple, rapid, and universal detection of pancreatic cancer-related miRNAs, which is significant for improving the accuracy of pancreatic cancer diagnosis. [ABSTRACT FROM AUTHOR]