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This research presents the synthesis and characterization of Cu-doped Fe 3 O 4 (Cu–Fe 3 O 4) nanoparticles as a magnetically recoverable and reusable detoxifying agent for the efficient and long-lasting neutralization of bacterial toxins. The nanoparticles were synthesized using the combustion synthesis method and characterized through SEM, XRD, BET, TGA, and VSM techniques. The detoxification potential of Cu–Fe 3 O 4 was compared with traditional formaldehyde (FA) in detoxifying epsilon toxin (ETx) from Clostridium perfringens Type D, the causative agent of enterotoxemia in ruminants. In vivo residual toxicity tests revealed that Cu–Fe 3 O 4 could detoxify ETx at a concentration of 2.0 mg mL−1 within 4 days at room temperature (RT) and 2 days at 37 °C, outperforming FA (12 and 6 days at RT and 37 °C, respectively). Characterization studies using dynamic light scattering (DLS) and circular dichroism (CD) highlighted lower conformational changes in Cu–Fe 3 O 4 -detoxified ETx compared to FA-detoxified ETx. Moreover, Cu–Fe 3 O 4 -detoxified ETx exhibited exceptional storage stability at 4 °C and RT for 6 months, maintaining an irreversible structure with no residual toxicity. The particles demonstrated remarkable reusability, with the ability to undergo five continuous detoxification batches. This study provides valuable insights into the development of an efficient and safe detoxifying agent, enabling the production of toxoids with a native-like structure. The magnetically recoverable and reusable nature of Cu–Fe 3 O 4 nanoparticles offers practical advantages for easy recovery and reuse in detoxification reactions. [Display omitted] • Cu-doped Fe 3 O 4 nanoparticles were synthesized and used for detoxifying ETx. • Cu–Fe 3 O 4 -detoxified ETx preserved almost its native ETx structure. • Durable toxoid without irreversible structure was achieved by Cu–Fe 3 O 4 in short time. • Cu–Fe 3 O 4 was a magnetically reusable detoxifying agent for detoxification. • Cu–Fe 3 O 4 -detoxified ETx led to the higher immune response. [ABSTRACT FROM AUTHOR]