The present invention provides a process for producing a biologically active botulinum neurotoxin comprising the following steps:(a) providing a culture medium, which is entirely free of animal-derived products or contains animal-derived products in an amount of less than 1 wt.%, inoculating the culture medium with Clostridium botulinum bacteria, and culturing the Clostridium botulinum bacteria until the optical density of the culture medium at 540 nm is 1.8 ± 1.0 AU；(b) transferring the culture medium having an optical density of 1.8 ± 1.0 AU at 540 nm into a fermentation medium, which is entirely free of animal-derived products or contains animal-derived products in an amount of less than 1 wt.%, and fermenting the Clostridium botulinum bacteria in the fermentation medium until a cellular autolysis phase is reached；(c) harvesting the fermentation medium by filtration；(d) concentrating the harvested fermentation medium and diluting the concentrated fermentation medium；(e) purifying the concentrated fermentation medium with an anion exchange column；(f) further purifying an eluate from the anion exchange column with a cation exchange column to obtain a purified eluate；(g) concentration and buffer exchange of the purified eluate； and(h) performing bioburden reduction filtration with the concentrated and buffer exchanged eluate；wherein no animal-derived protein is used to purify the botulinum neurotoxin.
