Cancer is a global burden with escalating incidence and mortality, often as a result of late diagnosis. Therefore, early detection, in order to improve prognosis and survival, remains a vital strategy for cancer management. A promising method for early diagnosis requires highly sensitive biomarkers and suitable technologies to detect them. Highly abundant in cells, microRNAs (miRNAs or miRs) play a key role as regulators of gene expression. A proportion of them are found circulating in biofluids, making them ideal non-invasive biomarkers for pathologies which aberrantly express miRNAs, such as cancer. Peptide Nucleic Acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridising to complementary nucleic acids with high affinity and specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesised to target specific miRNAs that were previously identified (and in some cases already validated) as potential diagnostic biomarkers for either Prostate Cancer (PCa) (miR-141 and miR-375) or Epithelial Ovarian Cancer (EOC) (miR-132). The sensing strategy is based on oligonucleotide-template reactions (OTRs) where only the miRNA of interest serves as a matrix to catalyse an otherwise highly unfavourable fluorogenic reaction. Validated in vitro using synthetic DNA and RNA oligonucleotides, these newly developed biosensors were then shown to detect endogenous concentrations of miRNA in human blood samples, without the need for any amplification step, and with minimal sample processing. This low-cost, quantitative and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost devices or assay kits. To investigate this further three platforms were explored where out PNA probes were compartmentalised into water-in-oil micro-droplets, embedded into permeable hydrogels or immobilised onto microplates. Low-cost and highly versatile, these platforms could pave the way for new point-of-care testing or public screening tools based on miRNA detection.