In this study, a novel fluorescence nanoprobe based on MIL-101 metal-organic frameworks embedding into the agarose hydrogel was fabricated using a hydrothermal technique and used for sensitive quantification of deferiprone in exhaled breath condensate (EBC) samples. The morphology and characterization of MIL-101/agarose nanocomposite hydrogel was studied by transmission electron microscopy, dynamic light scattering instrument, powder X-ray diffraction analysis, and Fourier transform infrared spectroscopy. The probe showed a reasonable fluorescence intensity quenching in the presence of deferiprone due to the interactions between iron centers in MIL-101 (Fe) and deferiprone, which likely formed non-fluorescent complexes. The proposed nanoprobe demonstrated a linear calibration curve in the range from 0.005 to 1.5 µg mL− 1 with a detection limit of 0.003 µg mL− 1. The intra- and inter-day precision for determinations of deferiprone with concentration of 1.0 µg mL− 1were 0.3% and 0.4% (n = 5), respectively. This method demonstrated high sensitivity and specificity towards deferiprone in the EBC samples and also presented a sensing platform with simplicity, convenience, fast implementation, and cost-effective in medical monitoring.