The morphological, physiological and biochemical characteristics of Saccharomyces boulardii were studied for the preparation of highly active yeast. The optimal condition of the flask culture including inoculum size, culture temperature, pH value, were investigated separately. The results showed that S.boulardii was asporogenous and had better heat tolerance and acid tolerance growing well at 37°C and pH 2.0. We also found that thermal death temperature was 55~56°C and S.boulardii is well ethanol-tolerant, for the highest concentration of ethanol that S.boulardii can tolerate was 20%. The optimal culture conditions in a flask culture was YEPD medium with 50ml/250ml medium volume, natural pH, 5% inoculum size, 32°C culture temperature, 160 r/min shaker speed, and 24 hours cultivation period. Under these conditions, suspension was diluted 10 times and the optical density at 600 nm was up to 0.85.