Uric acid (UA) is a crucial biomarker for various metabolic and renal disorders, making its accurate determination essential for clinical diagnostics and disease management. This study presents a novel fluorometric method for UA detection using a glutathione@cadmium/carbon dots/uricase system (GSH@Cd/CDs/uricase system), which offers enhanced sensitivity and selectivity compared to existing techniques. The method employs a cascade reaction mechanism initiated by uricase-catalyzed oxidation of UA to produce H₂O₂. This H₂O₂ subsequently oxidizes GSH to oxidized glutathione (GSSG), releasing cadmium ions from GSH complexes. The liberated Cd²⁺ ions interact with two types of carbon dots (CDs) in the system: blue-emitting CDs (BCDs) and red-emitting CDs (RCDs). This interaction triggers a dual fluorescence response, characterized by a decrease in fluorescence intensity at 400 nm (BCDs) and an increase at 610 nm (RCDs), enabling ratiometric detection of UA levels. Comprehensive characterization and mechanistic investigations were conducted using various optical and morphological techniques, including transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and fluorescence spectroscopy. The method exhibits outstanding analytical performance, characterized by strong linearity (R² = 0.9932), a broad detection range (0.05–7.0 μM), and a low detection limit of 0.015 μM. The method demonstrated excellent selectivity against common interfering substances and was successfully applied to human serum and urine samples with high recovery values (97.65 % to 99.20 %), highlighting its potential for clinical applications. This innovative approach combines the specificity of enzymatic recognition with the sensitivity of dual-emission fluorescence, offering a promising tool for accurate UA quantification in complex biological matrices.