Abstract: In this study,39 gram-positive strains with negative results of catalase tests were isolated from home-made sauerkraut juice in Gansu province. Six strains with more than 1% acid production rates were obtained by screening using titration method and identified as Lactobacillus genus by morphology,and LS-9 strain showed the highest acid production rate among the six strains. The optimum carbon source for LS-9 strain was glucose and the optimum nitrogen source was tryptone by single factor experiments. The optimum cultivation conditions were 20 g/L glucose and 12 g/L tryptone at 36℃ by orthogonal test. Temperature had the greatest effect on the acid production of fermentation followed by tryptone amount,and glucose amount had the least effect on the acid production of fermentation. Acid production rate was 1.74%±0.05% under optimum conditions. LS-9 strain shared high identity with Lactobacillus casei by 16S rRNA sequencing and phylogenetic tree analysis. The results of whole-fat milk fermentation showed that the curd formation time of LS-9 was roughly 6 h with acidity of 102.4°T and viable count of 1.25×10⁸ CFU/mL.