Abstract: In order to improve Lactobacillus casei KL1 of bile salt hydrolase which was isolated from Tibetan Kefir since fermentation broth number of the living bacterium, the author adopted the method of the three factors and three levels orthogonal experiments [L₉(3⁴)] with 5 L automatic fermentor, and studied the effect of different mediums and fermentation conditions on the living bacteria number of the starter culture. The results showed that under the condition of the fermentation temperature of 40℃, the inoculation quantity controlled in 3%, fermentation broth pH 6.5 and the fermentation time of 20 h, using the improved MRS culture medium, the living bacteria number of starter culture would be 8.2×10⁹ CFU/mL. Under the optimize fermentation conditions, the living bacteria number was 25.63 times as many as that before.