The invention provides nisin nanometer particles as well as a preparation method and application thereof, and belongs to the technical field of preparation of nanometer particles. The preparation method comprises the following steps of mixing nisin with an acid solution to obtain a nisin solution, wherein the pH value of the acid solution is 1-4; and dropwise adding pure water to the nisin solution, and performing stirring at the speed of 100-300rpm under the condition of 20-30 DEG C so as to obtain a nanometer particle solution. According to the method provided by the invention, an organic solvent being high in pollution to environment is prevented from being used, and the pure water being free from pollution and low in price is used for preparing the nanometer particles, so that the preparation cost can be greatly reduced, and the nanometer particles being high in bacterium-resisting activity and good in stability can be obtained.