<p>A dual-mode ratiometric fluorescent nanosensor was developed for ultrasensitive detection of glucose via simultaneous measuring pH and O₂ changes during enzymatic oxidation. The nanosensor integrates pH-sensitive fluorescent probe FITC and O₂-sensitive probe RuDP into hybrid nanoparticle core, with glucose oxidase (GOx) immobilized poly-L-lysine shell. During GOx-catalyzed reaction with glucose, the nanosensor gives rise of a decrease in pH-sensitive fluorescence due to acidification and an increase in O₂-sensitive fluorescence owing to oxygen depletion, enabling a dual-mode readout under single-excitation wavelength. The emission ratio between O₂-sensitive peak and pH-sensitive peak, employs signal amplification and results in high sensitivity, compared to single O₂ and pH testing. The ratiometric fluorescent signal exhibits a strong linear response in the 0–1 mM glucose range with a detection limit of 7.3 µM. The sensor shows robust performance and high reproducibility in DMEM and human sweat. In vivo imaging of zebrafish further confirms the photostability, and sensitivity of nanosensor, highlighting their utility for glucose detection. This study presents the first ratiometric fluorescence amplification strategy employing dual pH/O₂ testing for real-time glucose detection. The proposed nanosensor promotes non-invasive glucose detection in biomedical research and dual-mode ratiometric amplification strategies for monitoring metabolic targets.</p> Graphical abstract <p></p>