The necessity of sample pretreatment, the limited absorbance variation in the concentration range of calibration plot are the common restrictions of the colorimetric glucose sensors used with the complex samples. A calibration plot in a concentration range far below the common level of complex biological sample mostly involves to use a high dilution factor for finding the actual glucose level in the complex sample. Glucose oxidase immobilized-Au nanoparticle attached-magnetic SiO2 (GOx@Au@MagSiO(2)) microspheres were proposed as a new magnetic nanozyme, allowing one-pot and direct determination of glucose concentration in human whole blood without using a sample pretreatment and a high dilution factor. As an unusual approach which is not coherent with the common strategy followed for colorimetric determination of blood glucose level, a calibration plot based on the common range of human whole blood (i.e. 2.8-38.9 mM) is generated with the synthesized nanozyme. GOx was directly linked onto Au nanoparticles (Au NPs) immobilized on the magnetic SiO2 microspheres via a simple protocol, using the thiol groups of its cysteine residues, as a unique property of GOx's, for the first time. The activity of free GOx could be highly preserved by the selected immobilization protocol. GOx@Au@MagSiO(2) microspheres exhibited peroxidase-like activity due to Au NPs immobilized on the magnetic microspheres, together with the glucose oxidation ability originated from GOx linked to Au NPs. The nanozyme was efficiently used for one-pot, direct determination of glucose level in human whole blood in the concentration range of 50-700 mg/dL (i.e. 2.8-38.9 mM). (c) 2020 Elsevier B.V. All rights reserved.