The invention relates to a method used for extracting an antibacterial composition from limulus reagent production waste materials. The method comprises following steps: 1, raw material treatment is carried out, wherein a lower layer precipitate waste material obtained in centrifugation of an emulsified product in limulus reagent production is collected, and is taken as a raw material I, and discarded blood plasma in limulus reagent production is collected, and is taken as a raw material II; 2, extraction of a tachyplesin crude extracted solution is carried out, wherein acidolysis is carried out, pH is adjusted so as to remove a precipitate product, and thermal denaturation is carried out; 3, preparation of a hemocyanin crude extracted solution from blood plasma is carried out; 4, preparation of a SOD crude extracted solution is carried out; 5, 60 to 80 parts of the tachyplesin crude extracted solution,10 to 20 parts of the hemocyanin crude extracted solution, and 10 to 20 parts of the SOD crude extracted solution are mixed, pH value is adjusted to 4 to 5, and filtering is carried out so as to obtain the antibacterial composition. According to the method, the crude extracted solutions are prepared rapidly and conveniently at low cost; the antibacterial composition is capable of inhibiting growth of a plurality of kinds of bacteria and fungus; compared with conventional chemical antibacterial products, the antibacterial composition possesses following advantages: the antibacterial composition is safe, mild, and effective, no toxic or side effect is caused, and generation of drug resistance and drug allergy caused by conventional antibacterial products can be avoided.