Dysregulation of microRNA expression levels is closely associated with a variety of human diseases, and their rapid and sensitive quantification is essential to clinical diagnosis and therapy. Because of their poor sensitivity, conventional quantification methods are unable to detect low-abundance microRNAs. Alternatively, nucleic acid amplification approaches have been introduced to improve the detection sensitivity, but most of them involve complicated probe design and time-consuming procedures. Herein, we report a simple, rapid, and sensitive fluorescent method for label-free detection of low-abundance microRNAs based on isothermal helicase-dependent amplification. In this assay, the target microRNA may specifically hybridize with the 3'-terminus of the linear probe to form a DNA-microRNA heteroduplex, protecting the probes from exonuclease I digestion. The remaining probes may be subsequently amplified by helicase-dependent amplification, generating an ultrahigh fluorescence signal within 30 min. This assay is very sensitive with a low detection limit of 12.8 fM and exhibits a large dynamical range from 100 fM to 10 nM. Moreover, this assay can discriminate different microRNA family members, and it can be used to absolutely quantify endogenous microRNA of total RNA samples extracted from cancer cells, providing a powerful tool for biomedical research and clinical diagnostics.