Metal-organic frameworks (MOFs) as a peroxidase mimic have been integrated with glucose oxidase (GOx) to achieve one-step glucose detection. However, limited by the loading amount of GOx, the performances of the developed glucose sensing assays still remain to be further improved to meet sensing requirements in diverse biological samples. Herein, with Fe3+ as the metal ion and 2-amino-benzenedicarboxylic acid as a ligand, a fluorescent Fe-based organic framework (NH2-MIL-101) with peroxidase-like activity was synthesized. Due to the large specific surface area (791.75 m2 g-1), 68 μg mg-1 GOx could be immobilized through the amidation coupling reaction, and the product was designated GOx@NH2-MIL-101. With OPD as the substrate, Gox@NH2-MIL-101 achieved highly efficient biomimetic cascade catalysis for one-step glucose detection through an inner filter effect: upon reacting with glucose, GOx@NH2-MIL-101 catalytically oxidized glucose using dissolved O2, and the produced H2O2 concurrently oxidized o-phenylenediamine (OPD) to oxidized OPD (oxOPD), accompanied by the fluorescence of GOx@NH2-MIL-101 at 456 nm being quenched and that of oxOPD at 565 nm being enhanced. With the fluorescent ratio F565/F456 used as a readout signal, a wide linear range of 0.1-600 μM was obtained, and the detection limit was 0.0428 μM. Based on the excellent selectivity and high stability of GOx@NH2-MIL-101, the developed assay was successfully applied to glucose detection in human serum and saliva, presenting potential applications in diverse biological samples and even medical diagnosis.