The invention relates to a preparation method of a natural source plant antibiotic and application thereof. The natural source plant antibiotic is prepared from kernels or shells of fructus crataegi, waxberries, wild jujubes, oranges, walnuts, eriobotrya japonica, fructus jujubae, apricot, peaches, plums, pomelo, pine nuts and filberts which are used as raw materials. The natural source plant antibiotic is wide in antibacterial spectrum and obvious in inactivation effect on virus, fungus, and bacteria, is safe and stable, has the functions of accelerating healing of mucosa wound surfaces, repairing pathological tissue and enhancing natural defense functions, and is capable of effectively preventing and treating harm of pathogenic microorganisms to the human body; meanwhile, the natural source plant antibiotic can be widely applied to multiple fields closely related to the human living environment such as disease prevention, senescence resistance and healthcare, skincare, sanitation supplies, cosmetics, daily chemical products, air sterilization, foods, medicines, feed additives, food additives, deodorants, soil conditioners, control and prevention of pests and improvement of yield of vegetables and crops; and the natural source plant antibiotic has extensive utilization value and development prospect.