The invention discloses a glucose detection reagent magnetic nano-microsphere and a preparation method thereof. The formula includes: 26-42 parts of gelatin, 18-34 parts of quaternized chitosan, 2-6 parts of lecithin, seaweed 16-28 parts of sodium bicarbonate, 0.6-1.2 parts of magnetic nanoparticles, 80-160 parts of absolute ethanol, 20-30 parts of glucose oxidase, 10-20 parts of dehydrogenase, 10-20 parts of o-benzidine, surface 9-15 parts of active agent, 60-120 parts of Tris buffer; the preparation method wraps each reagent individually with a microsphere polymer shell substrate, so that the reagent can maintain the stability of the reagent even under harsh transportation conditions , and enzyme activity, prolong the storage time of detection reagents, and improve detection accuracy.