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A novel multi-enzyme co-immobilized nanoparticle was prepared by the bis-aryl hydrazone conjugation strategy for enhancing the overall reaction efficiency and specificity. It can be seen that the molar substitution ratios of succinimidyl 6-hydrazinonicotinamid acetone hydrazone or succinimidyl 4-formylbenzoate to glucose oxidase (GOX) or horseradish peroxidase (HRP) increased with the concentration of the linking reagents. The amount of the immobilized conjugates on the support was measured to be 22.8 ± 1.6 mg/g-particle, and it meant that more than 90% of the GOX–HRP conjugates were successfully attached onto the polystyrene (PS) nanoparticles. Moreover, the immobilized bi-enzymes conjugate on the PS nanoparticles increased about 1.6-fold compared with that of free enzymes.