Nanozymes have attracted attention owing to their distinct catalytic capabilities and potential applications, being advantageous compared to natural enzymes in terms of storage and cost efficiency. In this study, we investigated the enzymatic properties of iron oxide nanoclusters (IOCs) formed through the clustering of small nanoparticles. Our findings reveal that the enzymatic activity of IOCs is enhanced as their size increases. Additionally, we demonstrated that the size of the unit particles within IOCs is highly dependent on the nucleation environment, which is a crucial factor in determining the overall size of the IOCs. Importantly, the surface area of IOCs is more closely related to the size of the individual unit particles rather than the entire cluster. Smaller unit particle sizes within IOCs resulted in an increase in nanocluster size, thereby augmenting the specific surface area. The optimal IOC exhibited superior stability under various conditions and a broader range of reactivity compared to natural enzymes, making it a promising probe material for point-of-care tests across diverse environments. Furthermore, its effectiveness as a glucose detection probe was demonstrated, highlighting its potential for practical applications. The remarkable enzyme-like efficacy of IOCs not only enhances their utility in on-site detection technologies but also establishes them as a versatile detection probe.