The invention belongs to the technical field of microbes, and in particular relates to an antimicrobial compound and a fermenting preparation method and application thereof. The molecular weight of the antimicrobial compound of the invention is 445, the molecular formula of the antimicrobial compound is C16H23N5O10, the chemical name of the antimicrobial compound is 1-uracil-4-sarkosyl-serylamido-1,4-dideoxy-beta-D-glucopyranosyl acid, and the antimicrobial compound belongs to uracil hexose dipeptide and is named xin'aomycin. The preparation method adopts actinomycetes Streptomyces noursei and genetically improved strains thereof, and comprises the following steps: culturing in a primary liquid culture medium as a seed liquid; inoculating the cultured seed liquid to a secondary liquid culture medium B for culturing, and carrying out fed-batch fermentation culture of fed-batch liquid C; and after fermentation is finished, separating and purifying the structural compound from the fermentation culture liquid. The antimicrobial compound is used for manufacturing pesticide or medicine products. The invention has the characteristics of high yield, simple process and easy industrialization.