We present and demonstrate a novel assay for the detection and quantification of microRNA (miRNA) that leverages isotachophoresis (ITP) and molecular beacon (MB) hybridization. We use ITP to selectively preconcentrate miRNA from total RNA. We simultaneously focus MBs and use the ITP zone as a 10 pL reactor with active mixing where MBs fluoresce upon hybridization to target miRNA. To increase both sensitivity and selectivity, we leverage a multistage ITP strategy composed of three discrete regions of different concentrations of denaturant, sieving matrix, and magnesium chloride. We show that ITP hybridization is specific and selective to the miRNA target. We demonstrate ITP hybridization of miRNA in a biologically relevant case by detecting and quantifying miR-122 in human kidney and liver. ITP hybridization is a cheap, simple, high-speed, and amplification-free miRNA profiling method which requires small amounts (order 100 ng) of sample. The technique therefore represents an attractive alternative to PCR or Northern blot for miRNAs.