Screening lower high cholesterol and secretion of bile salt hydrolase (BSH) from yeast strains, and study the environmental factors for increasing the activity of BSH, then investigate the mechanism of cholesterol lowering. We obtained two yeasts to lower cholesterol by high-throughput screening technology and o-phthalaldehyde : strain K1 and strain M3 are Kluyveromyces marxianus, are the highest rate of cholesterol-reducing strains by far, the lower cholesterol rates were 68.14% ~ 70.34% , 80.51% ~ 99.12%. The two yeasts can secretion BSH in the metabolic process with the Oxford cup method, and the BSH activity is proportional to the rates of cholesterol-reducing. Strain M3 has a higher BSH production capacity than strain K1, and high cholesterol-lowering effect. That indicating efficient yeast strains to lower cholesterol production is due to their abundant or high activity BSH. The BSH secretion of strain K1 and strain M3 demonstrated a key enzyme in cholesterol-lowering effect for the first time. BSH can hydrolyze conjugated bile salt and free bile salt, which can precipitate with cholesterol state, to reduce the effect of cholesterol. To aim directly at 4 major factors can affect the synthesis of BSH, the fermentation conditions of strain M3 producing BSH highly were determined with four factors and three levels［L9(34)］orthogonal experiment: fermentation temperature is 32 °C, fermentation time is 24h, the initial pH of medium is 6.0, inoculums is 3%. The activity of BSH secreted by Strain M3 increased 50% in the optimization of fermentation conditions. Strain K1 and M3 fermented milk with excellent performance, we could research and develop functional fermented dairy products and micro-ecology preparation, by using the characteristics of efficient to lower cholesterol and complementary advantages of lactose fermentation performance of the two strains.