The invention discloses a lactobacillus rhamnosus culture medium. The lactobacillus rhamnosus culture medium and culture method has a basic culture medium and an optimized culture medium, wherein the basic culture medium is prepared from the following components: yeast peptone, glucose, yeast extract, sodium acetate, magnesium sulfate, tween 80, isomaltooligosaccharide, potassium dihydrogen phosphate and the balance of sterile water, and the pH value is 6.20 to 6.80; and the optimized culture medium is prepared from the following components: yeast peptone, glucose, yeast extract, sodium acetate, magnesium sulfate, tween 80, isomaltooligosaccharide, potassium dihydrogen phosphate and the balance the sterile water, and the pH value is 6.20 to 6.80. The culture medium is suitable for proliferation culture of lactobacillus rhamnosus. By utilizing the method for culturing the lactobacillus rhamnosus by virtue of the culture medium, not only can the culture of the lactobacillus rhamnosus be effectively proliferated, but also the industrialized mass production can be facilitated.