In order to apply excellent lactic acid bacteria strains from pastoral area from Gansu province to production of direct vat set starter(DVS),Lactococcus lactis subspecies(HM 598683),Lactobacillus acidophilus(HM 598684) and Lactobacillus casei(HM 598685) from Gansu province were implemented in mixed fermentation by 1: 0:0,0:1:0,0:0:1,1:1:0,1:2:0,2:1:0,1:0:1,1:0:2,2:0:1,1:1:1,1:2:1,2:1:1,1:1:2,the formula of mixed fermentation was determined through sensory attributes and biochemical characteristics.And the enrichment medium of mixed fermentation was also optimized.The results showed that curd time was minimum with 8.5 h,the colony forming unit(CFU) was maximum with 7.90×10~8 CFU/mL and flavour and shape in sensory attributes was best when the ratio of strains was 1:1:1.According to methylene blue test,the activity of lactic acid bacteria was highest when trypsin was selected for hydrolyzing milk protein and the CFU was up to 2.93×10~8 CFU/mL.The optimum multiplication factors were determined by using L_9(3~4) orthogonal design with the CFU up to 2.11×10~9CFU/ mL,which were yeast powder 1%,fructose 0.5%,Tween 0.2%,sodium acetate 0.5%.The rank of effect of multiplication factors on CFU was yeast powder > sodium acetate > fructose > Tween.