The invention relates to a culture medium for culturing chicken's normal intestinal bacteria flora and a preparation method of the culture medium, and belongs to the technical field of microbiological culture medium preparation. A pH value of the culture medium is 6.8-7.1; the culture medium is mainly prepared from the following components: bean sprout liquid, fish meal peptone, cysteine hydrochloride, soybean peptone, vitamin B1, beef extract, beef liver extract powder, yeast extract powder, soluble starch, glucose, dipotassium phosphate and magnesium sulfate. The culture medium disclosed by the invention is in a liquid state and can be used for determining the content of effective viable bacteria, the content of probiotics, a proportion of bacillus to coccus, and a proportion of positive bacteria to negative bacteria in the bacterial flora obtained by culture as well as a biological activity expiration date of probiotics by virtue of the composition and the content of each component. Therefore, the bacterial flora which is almost consistent with the chicken's normal intestinal bacteria flora in the proportion of bacillus to coccus and the proportion of positive bacteria to negative bacteria can be cultured by adopting the components and the proportions of the culture medium.