<p>Glucose is a critical energy source for human cells, and maintaining stable glucose levels is essential for normal physiological functions. Abnormal glucose levels can lead to health issues such as diabetes, obesity, and hypoglycemia, highlighting the need for precise and rapid glucose detection methods for clinical diagnostics and health management. In this study, a novel ratiometric fluorescent probe was constructed for glucose detection. Using glucose oxidase (GOx) and horseradish peroxidase (HRP) as enzymatic mediators, the probe employs orange-emitting carbon dots (oCDs) as the detection signal and red-emitting quantum dots (rQDs) as the reference signal. Glucose is oxidized by GOx to produce hydrogen peroxide (H₂O₂), which subsequently generates hydroxyl radicals (·OH) under HRP catalysis. The ·OH interacts electrostatically with oCDs, forming non-fluorescent complexes and quenching the oCDs fluorescence. Glucose concentration is quantified by monitoring the fluorescence intensity ratio (<i>I</i>₅₉₀/<i>I</i>₇₁₅) between oCDs and rQDs. The probe has a limit of detection (LOD) of 0.47 µM and a limit of quantification (LOQ) of 1.58 µM. After methodological validation, it has been successfully applied to the detection of glucose in human serum. This study provides a solid basis for accurate glucose monitoring and holds potential for the early diagnosis and treatment of metabolic diseases such as diabetes.</p>