Abstract: In order to improve the ability of cholesterol degradation and the number of viable bacteria, the study optimized the fermentation conditions of mixed strains, aiming at obtaining high efficient cholesterol-degrading strains and providing the basis for the application of lactic acid bacteria. Four strains of lactic acid bacteria with auxiliary cholesterol-lowering activity were selected and mixed with 1∶1∶1∶1 mixture. The single factor experiment and response surface analysis were used to study the effects of cholesterol degradation rate and viable count.Conditions were optimized.The optimum fermentation conditions were as follows: lactose 24.19 g/L, tryptone 8.41 g/L, culture temperature 28.28 ℃, culture time 30 h, inoculation amount 5.24%.After optimization, the degradation rate of cholesterol in the mixed strains increased from 39.56% to 53.43%, increased by 35.06%.The totol colonies was increased from 8.87 lg CFU/m L to 9.63 lg CFU/m L. The viable count was an order of magnitude higher than that before optimization.