The invention relates to a culture medium for culturing a living biological medicine and application of the culture medium. The culture medium for culturing the living biological medicine is prepared from peptone, beef extract, lactulose, mannose oligomer, raffinose, yeast extract, diammonium hydrogen citrate, K2HPO4, MgSO4, MnSO4, Tween-80 and L-cysteine. The living biological medicine comprises any one or a combination of at least two of lactobacillus reuteri, lactobacillus paracasei, lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium longum, pediococcus pentosaceus, lactobacillus crispatus, lactobacillus jensenii or lactobacillus gasseri. The culture medium has an unexpected promoting effect on growth, proliferation, metabolism and efficacy exertion of living biological medicines. The efficacy is such as prevention or treatment of gastrointestinal diseases, immune diseases, cancers, vaginitis and other diseases.