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Understanding the structure−activity correlation and reaction mechanism of the catalytic process in an acetic acid−sodium acetate (HAc-NaAc) buffer environment is crucial for the design of efficient nanozymes. Here, we first reported a lattice restructuration of Au-LaNiO 3−δ nanofibers (NFs) after acidification with the HAc-NaAc buffer to show a significantly enhanced oxidase-like property. Surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT) calculation confirm the direct evidence for the formation of specific enhanced intermediate O−O species after acidification, indicating that the insertion of the carboxyl group in the A-Au/LaNiO 3−δ NFs plays crucial roles in both producing vacancies in HAc-NaAc solution from its dissociation during the catalytic process and the protection of the vacancies, which can be directly interacted with oxygen in the environment to produce O−O species, realizing the enhanced oxidation of substrate molecules. The insertion of the carboxyl group increased the oxidase-like catalytic activity by 2.38 times and the SERS activity by 5.27 times. This strategy offers a way to construct an efficient nanozyme-linked immunosorbent assay system for the diagnosis of cancer through the highly sensitive SERS identification of exosomes.