The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nanostructures for precision disease diagnosis remains unexplored. Particularly, DNAtemplated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks (CuNBs) on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance (quantitative reverse transcriptionpolymerase chain reaction (PCR) validation). The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.