The invention relates to an antibiotic, and a preparation method and an application thereof. The preparation method comprises the following steps: activating Lactococcal lactis ATCC 11454, picking single colony, culturing, centrifuging to remove bacterial strains to obtain a fermentation broth, adjusting the pH value of the fermentation broth to 2, carrying out rotary evaporating concentration, adjusting the pH value of the obtained concentrate to an initial value, centrifuging, taking the obtained supernatant, and filtering to obtain a fermentation concentrate; and mixing the fermentation concentrate with a PBS buffering solution, adding a double water phase system formed by Triton X-114 and MgSO4, centrifuging to realize phase balance of double water phases, taking the obtained upper phase, and diluting the upper phase by using the PBS buffering solution to prepare the antibiotic containing nisin and Triton X-114. The antibiotic has a substantial bacteriostasis effect on Gram-positive bacteria. The antibiotic includes nisin and Triton X-114, and the combination of the nisin and the Triton X-114 makes the antibiotic have high titer. The preparation method has the advantages of simple extraction process and low cost, and is suitable for intermittent and large-scale production and processing of high-tilter and high-yield antibiotics.