Artificial nanozymes are gaining prominence as robust alternatives to natural enzymes for catalytic and sensing applications, yet their use is often hindered by poor colloidal stability, inefficient electron transfer, and low catalytic activity under harsh conditions. To address these limitations, we developed a chitosan-imide-functionalized Fe₃O₄ nanocomposite (FeNp-CSNI) via sequential hydrothermal synthesis, polymer grafting, and interfacial assembly. Comprehensive material characterization was carried out using XRD, FTIR, BET, TEM, EIS, and DFT analyses. FeNp-CSNI exhibited a worm-like mesoporous morphology with high surface area, reduced bandgap, and significantly lower charge transfer resistance, suggesting improved electron mobility and catalytic efficiency. The peroxidase-like catalytic activity was assessed by OPD oxidation in the presence of H₂O₂, where hydroxyl radicals were confirmed as the dominant reactive species. Under visible-light irradiation, FeNp-CSNI achieved >95% degradation of crystal violet, demonstrating strong photocatalytic activity. The nanozyme retained ~97% of its catalytic performance over five reuse cycles, indicating high stability. Furthermore, real-sample testing showed accurate and efficient detection of H₂O₂ in milk, with excellent recovery rates, highlighting the nanozyme’s practical potential in biosensing. This work establishes that interfacial engineering of Fe₃O₄ nanoparticles with chitosan-imide substantially enhances both catalytic performance and environmental durability. The FeNp-CSNI nanozyme offers a multifunctional and sustainable platform for biosensing, environmental remediation, and photocatalytic applications, paving the way for future nanozyme-based technologies.