Detecting nucleic acid biomarkers is essential for early cancer diagnosis and monitoring. Traditional methods (e.g., exponential isothermal amplification (EXPAR)) adopt DNA polymerases and endonucleases for nucleic acid amplification and analysis, but they are limited by the dependence on specific sequences and nonspecific amplification. Here, we establish a programmable and universal nucleic acid diagnostic platform named Functional Junction Molecule Unveil (FJMU) for microRNAs detection. FJMU incorporates Argonaute (Ago) nuclease and DNA polymerase to accomplish nucleic acid detection in a single reaction. It utilized a specially designed repetitive sequence template to achieve synchronized dual-cycling amplification of biomarkers. The FJMU method eliminates the reliance on restriction endonucleases such as Nt.BstNBI used in EXPAR. FJMU showed high specificity and stability in detecting colorectal cancer-related microRNAs. A comparison of the detection results between the FJMU method and the reverse transcription-polymerase chain reaction (RT-PCR) for clinical serum samples suggests that the FJMU method is potentially feasible for clinical applications. FJMU provided a programmable platform for detecting microRNAs and further explored the Ago-mediated nucleic acid detection technology.