<p>The accurate detection of double-stranded DNA (dsDNA) cancer biomarkers remains a critical challenge in biosensing technologies, as native strand pairing hampers probe hybridization. While photoelectrochemical (PEC) biosensors hold promise for rapid and cost-effective nucleic acid diagnostics, their application has largely been confined to synthetic single-stranded DNA (ssDNA), limiting their clinical applicability. Here, we introduce an enzyme-free PEC biosensing strategy that directly detects DNA targets within their native double-stranded form by integrating locked nucleic acid (LNA) probes into a singlet oxygen (¹O₂)-based PEC assay. Using <i>KRAS</i> G12C as a dsDNA model, systematic probe engineering identified a 17-nucleotide capture probe with three LNA modifications flanking the mutation site as the optimal design for the capture probe. The biosensor achieved a limit of detection of 20 pM for <i>KRAS</i>-mutated dsDNA, a discrimination efficiency of 92.9% against <i>KRAS</i> wild-type (WT) sequences, and reliable detection of 1% variant allele frequency (VAF) in a WT background, closely reflecting clinical scenarios. This study represents the first use of LNA-modified probes in PEC biosensing and offers a robust strategy for enzyme-free detection of dsDNA targets, with broad potential for precision oncology and point-of-care applications.</p> Graphical Abstract <p></p>