The invention discloses a culture medium for effectively prolonging the survival time of lactic acid bacteria and a preparation method of the culture medium. The culture medium for effectively prolonging the survival time of the lactic acid bacteria is prepared by taking peptone, yeast extract powder, mannan oligosaccharide, skim milk powder, defatted soybean meal, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, dried corn steep liquor powder, a compound additive, sodium dihydrogen phosphate, sodium citrate, citric acid, sodium acetate, Tween-80 and saccharomyces cerevisiae as raw materials; in addition, the compound additive is a mixture of sodium carboxymethylcellulose, sodium alginate, bone meal, royal jelly acid, guar gum and xanthan gum. According to the culture medium for effectively prolonging the survival time of the lactic acid bacteria, the functions of all the raw materials are mutually cooperative, and higher variable bacteria quantity of the lactic acid bacteria can be kept at normal temperature for a long time; in addition, the culture medium has the advantages of simplicity and convenience in operation, easily-obtained raw materials, low cost and high homogenization degree of a product; the lactic acid bacteria is enabled to be more stable in property and higher in biological activity and can be stored for a longer time.