Abstract: Method for preparing botulinum neurotoxin and nanoparticle thereof are provided. The method includes fermenting bacteria Clostridium botulinum in a fermentation media free of animal-derived ingredients and contacting the fermentation media with an anion exchange media slurry and obtaining a supernatant including the botulinum neurotoxin by centrifugation. The method further includes dialyzing the supernatant and collecting a dialyzed solution including the botulinum neurotoxin, contacting the dialyzed solution with an anion exchange chromatography column, contacting an elute collected from the anion exchange chromatography column with a cation exchange chromatography column, and collecting an elute. The nanoparticle includes multi-layer including an innermost water phase core including biomolecules encapsulated by an oil phase layer, thereby forming a water-in-oil structure, water phase layers; oil phase layers; and an outmost cream layer.