Abstract: MicroRNAs (miRNAs) are small oligonucleotides (18–25 bases), biologically relevant for epigenetic regulation of key processes, particularly in association with cancer. Research effort has therefore been directed towards the monitoring and detection of miRNAs to progress (early) cancer diagnoses. Traditional detection strategies for miRNAs are expensive, with a lengthy time-to-result. In this study we develop an oligonucleotide-based assay using electrochemistry for the specific, selective and sensitive detection of a circulating miRNA (miR-141) associated with prostate cancer. In the assay, the excitation and readout of the signal are independent: an electrochemical stimulation followed by an optical readout. A ‘sandwich’ approach is incorporated, consisting of a biotinylated capture probe immobilised on streptavidin-functionalised surfaces and a detection probe labelled with digoxigenin. We show that the assay allows the detection of miR-141 in human serum, even in the presence of other miRNAs, with a LOD of 0.25 pM. The developed electrochemiluminescent assay has, therefore, the potential for efficient universal oligonucleotide target detection via the redesign of capture and detection probes.