Abstract Magnetite (Fe 3 O 4 ) nanoparticles, widely recognized as inorganic nanozymes due to their enzyme‐like catalytic activity, are emerging as effective heterogeneous catalysts for Fenton‐like reactions, in which lattice iron activates hydrogen peroxide (H 2 O 2 ) to generate reactive oxygen species. While hydroxyl radicals (•OH) are generally considered the primary reactive species, the underlying mechanism—particularly the possible involvement of a high‐valent ferryl intermediate (Fe 4+ ═O)—remains under debate. Here, surface‐specific spectroscopy with density functional theory (DFT) calculations is used to elucidate the mechanism of H 2 O 2 activation on Fe 3 O 4 (001) surfaces. It is found that •OH production is driven by electron transfer from subsurface Fe 2 ⁺ centers to adsorbed H 2 O 2 , accompanied by the transient formation of a ferryl species. Moreover, interfacial water plays an active role in modulating surface reactivity and stabilizing key reaction intermediates. These findings clarify the origin of radical formation in Fe 3 O 4 nanozymes and offer mechanistic insight to guide the rational design of next‐generation oxide‐based catalysts for environmental and biomedical applications.