The invention discloses a preparation method and application of an antimicrobial composition containing ε-polylysine. It consists of 1-20% of ε-polylysine, 5-35% of other antimicrobial agents, and 60-90% of solvent and synergist. The antimicrobial agent is one of caprylyl hydroxamic acid, levulinic acid, sodium levulinate, p-hydroxyacetophenone, ethyl lauroyl arginate hydrochloride, tropolone, and octenidine one or a mixture of two. Solvents and builders are 1,2-propylene glycol, methyl propylene glycol, 1,3-propylene glycol, 1,3-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2 A mixture of one or at least two of ‑caprylyl glycol, 1,2‑decanediol, polyethylene glycol, ethylhexylglycerin, propylene glycol carbonate, and water. The composition is water-soluble, convenient to add and use, and widely used in the fields of cosmetics, wet wipes, facial masks, washing and the like.