The invention discloses a culture medium for selectively separating and culturing group B streptococci and a preparation method thereof. Culture supernatant fluid of group B streptococci at the logarithmic phase and an antibiotic combination are added to a group B streptococcus selective developing basic culture medium. The culture supernatant fluid contains quorum sensing substances capable of accelerating the growth of bacteria and facilitating the speed of color development reaction and color development intensity, thereby greatly shortening the detection time; and the antibiotic combination comprises amethopterin, colistin sulfate and metronidazole, which can inhibit the growth of mixed bacterium, except the group B streptococci, thereby achieving a purpose of selectively separating and culturing the group B streptococci. When the culture medium is used for detecting the group B streptococci, the characteristics of high specificity and sensitivity, convenience in observation and the like are provided, and the culture medium can be effectively applied to rapid detection to clinic samples.