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Nanozymes have gained prominence for their utility in biosensing and disease diagnostics. However, challenges arise from complex sample matrices and nonspecific enzyme activities that contribute to false signals. This study introduces multifunctional platinum nanoclusters (Pt NCs) exhibiting peroxidase-like (POD-like), oxidase-like (OXD-like), and laccase-like activities tailored for enhanced biosensing capabilities. By adjusting pH, we optimized the conditions to achieve distinct POD-like and OXD-like responses, thereby reducing background signals and improving detection accuracy. The addition of ATP further amplified the POD-like activity while minimizing interference from OXD-like activity. This combined strategy substantially enhanced biomarker detection selectivity, demonstrated through glucose detection in human serum samples. Moreover, thiol inhibition of laccase-like activity in Pt NCs was leveraged for thiol-based antioxidant assessment, revealing their application in quantifying total antioxidant capacity (TAC) in human liver cancer cells, accounting for 44 % of TAC. The Pt NCs demonstrated robust sensitivity and reusability, offering a novel multi-enzyme nanomaterial with potential for precise and interference-free biosensing applications. These findings contribute to the development of advanced nanozyme-based biosensors, addressing specificity regulation challenges and expanding their practical application in biosensing and disease diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)