DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide build-ing blocks to provide sequence-specific, PCR-free fluorescent detection of un-labelled nucleic acid sequences. We discrimi-nated at picomolar detection limits (<7 pM) ‘perfect-match’ from mismatched sequences, down to a single nucleotide muta-tion, buried within longer lengths of target. Detailed characterisation by NMR, TEM, mass spectrometry and rheology pro-vided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sen-sitivity and detection limit. We discuss the generic design, which is based on a highly-predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g. SNPs) and RNA (messenger, micro and viral genomic RNA).