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A novel magnetic nanozyme Fe3O4@MXene-Au nanocomposite, which possessed higher peroxidase-like activity than that of Fe3O4 nanoparticles and Fe3O4@MXene nanocomposites, was developed. The outstanding magnetic properties of the nanozyme endowed it with the ability of simple and rapid separation, achieving great recyclability. Based on Fe3O4@MXene-Au nanocomposites and glucose oxidase (Glu Ox), a highly selective colorimetric biosensor for glucose detection was developed. Fe3O4@MXene-Au nanocomposites can catalyze H2O2 produced from glucose catalyzed by glucose oxidase to ·OH and oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) with a significant absorbance at 652 nm. The linear range of glucose was 0–1.4 mM under optimal conditions, with a limit of detection (LOD) of 0.11 mM. Glucose in human whole blood was successfully detected with satisfactory recoveries. Furthermore, a facile agarose hydrogel detection platform was designed. With smartphone software, glucose detection can be realized by the agarose hydrogel platform, demonstrating the potential in on-site and visual detection of glucose. [ABSTRACT FROM AUTHOR]