<p>Chronic hepatitis B virus (HBV) infection remains a significant global health challenge. Accurate quantification of HBV relaxed circular DNA (rcDNA) is critical for monitoring viral replication and assessing disease progression. However, conventional isothermal amplification methods are often compromised by limited specificity. To address these limitations, we engineered a toehold-triggered switchable DNA three-way junction (3WJ) protective nanoprobe that synergizes RNase H-assisted target recycling amplification (RATRA) with toehold-mediated strand displacement (TMSD). Leveraging the programmability of DNA nanotechnology, we constructed a rigid 3WJ scaffold featuring a “protective” nanoscale cavity at the branching point. This architecture effectively sequesters the ribonucleotides via steric hindrance, preventing non-specific enzymatic cleavage in the absence of the target. Upon specific recognition of the HBV rcDNA gap region, the nanoprobe undergoes a precise conformational switch, initiating a cyclic cleavage and signaling cascade. Governed by a dual-verification mechanism, the assay demonstrates exceptional specificity, enabling the discrimination of single-base mutations. Under optimized conditions, the method achieved a limit of detection (LOD) of 87.28 fM with a broad linear dynamic range. Validation in clinical serum samples revealed strong concordance with gold-standard quantitative real-time PCR (qRT-PCR). Consequently, this work presents a robust, modular, and cost-effective platform for point-of-care viral diagnostics.</p> Graphical Abstract <p></p>