The aim of this work is to develop the synthesis of the magnetically separable biocatalyst for enzymatic oxidation of D-glucose to D-gluconic acid with high product yields. The biocatalyst support is based on magnetite particles (MPs) synthesized by coprecipitation and coated with the amino- silica layer to facilitate further functionalization. This functionalization involves the attachment of the glutaric dialdehyde linker followed by the covalent attachment of glucose oxidase (GOx, an enzyme of oxidoreductase group) via its amino groups. TEM, XRD, and magnetic measurements were performed for initial MPs and GOx biocatalyst. The biocatalyst activity was studied in the oxidation reaction of D-glucose to D-gluconic acid. The biocatalyst synthesis proposed in this work allowed to create a highly effective system. It was found that GOx immobilized on the modified MPs was the most active biocatalytic system in comparison with other inorganic support and showing 88% of gluconic acid yield.