The invention discloses a culture method of Tibetan kefir grains. The method comprises the following steps of 1, culture medium configuration, 2, packaging, 3, sterilization, 4, cooling, 5, inoculation, 6, fungus culture, 7, growth promoting, 8, fruiting, 9, harvesting and 10, secondary harvesting. According to the scheme, adopted raw materials are free of pollution and have rich nutrition, and no chemical fertilizer or pesticide or sanitizer or other chemical preparation is used in the culture process, so that damage caused by chemical preparations to the human body is avoided, the environmental pollution problem is solved on the premise of ensuring the heath of people, the nutrition value of the Tibetan kefir grains is ensured, the fruiting conversion rate is obviously improved, and then the fruiting amount is improved.