We developed exponentially amplified rolling circle amplification with CRISPR–Cas12a as a one-pot, isothermal assay for microRNA detection. This method has single-digit femtomolar sensitivity and single-nucleotide specificity, and can be deployed for point-of-care testing. The assay has been adapted for the microRNA profiling of extracellular vesicles, which is used in the diagnosis of pancreatic cancer.