<span class="paragraphSection"><div class="boxTitle">Abstract</div>G-quadruplex DNA (G4-DNA), a noncanonical tetrahelical nucleic acid structure stabilized by stacked G-quartets via Hoogsteen hydrogen bonding, plays critical roles in genomic regulation and disease pathogenesis. Current methodologies for detecting these structures face limitations in specificity, spatiotemporal resolution, and live-cell applicability. To address these challenges, we engineered G4-Flame, a genetically encoded fluorescent biosensor utilizing circularly permuted fluorescent protein technology. By strategically positioning a G4-specific binding domain proximal to the fluorophore of circularly permuted YFP (cpYFP), G4-Flame achieves real-time, high-resolution visualization of G4-DNA dynamics in living systems, with specificity across diverse G4 conformations. Experimental validation revealed distinct spatiotemporal patterns of G4-DNA during the cell cycle: nuclear G4-DNA levels peaked during the S phase, while mitochondrial G4-DNA was found to suppress the expression of mitochondrial-encoded genes. Clinically, serum analysis revealed significantly elevated G4-DNA levels in cancer patients compared to healthy controls. This work establishes G4-Flame as a transformative tool for investigating G4-DNA spatiotemporal regulation and advances its potential as a biomarker for early cancer detection, bridging fundamental research with clinical translation.</span>