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Background: Neuraminidase is a pathogenic protein of the avian influenza virus. Previous studies have shown that silibinin has the potential to inhibit neuraminidase activity. Objective: This study aims to explore the interaction between silibinin and neuraminidase and the effect of silibinin on the structure and activity of neuraminidase. Methods: In this study, two-dimensional fluorescence spectrum, three-dimensional fluorescence spectrometry, Uv-vis spectroscopy, and circular dichroism analysis were used. Results: Silibinin alters the secondary structure of neuraminidase and inhibits the activity of neuraminidase. Conclusion: Silibinin can interact with neuraminidase and inhibit its activity.