The present disclosure relates to a method for preparing an antimicrobial complex strain from kefir, which includes: 1) providing and, optionally filtering, kefir or kefir whey; 2) introducing a fermentable sugar into the kefir or kefir whey, wherein based on the weight of the kefir or kefir whey, at least a saturation amount of said fermentable sugar is used, and allowing curd to gather at the top, so as to form a pre-fermentation composition; and 3) enabling the pre-fermentation composition to ferment until a microbial membrane that is capable of growth develops at the top and a whey liquid forms underneath, and the whey liquid demonstrates antimicrobial activity and has a pH of about 3.0, wherein during the fermentation, if needed, further adding said kefir or kefir whey and/or said fermentable sugar.