Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.