The invention discloses a preparation method and application of an epsilon-polylysine-containing antimicrobial composition. The epsilon-polylysine-containing antimicrobial composition comprises the following components of 1 to 20% of epsilon-polylysine, 5 to 35% of other antimicrobial agent, and 60 to 90% of solvent and synergistic additive, wherein the antimicrobial agent is a mixture of one or two of caprylhydroximic acid, levulinic acid, sodium levulinic acid, p-hydroxyacetophenone, ethyl lauroyl arginate, tropolone and octenidine; the solvent and synergistic additive are mixture of water and one or at least two of 1,2-propanediol, methylpropanediol, 1,3-propanediol, 1,3-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-octanediol, 1,2-decanediol, polyethylene glycol, ethylhexylglycerinand propanediol carbonate. The epsilon-polylysine-containing antimicrobial composition can be dissolved into water, be convenient in adding and use, and can be widely applied to the fields of cosmetics, wet tissues, masks, washing and the like.