In the riboflavin fermentation by recombinant Bacillus subtilis 24/pMX45,The yeast powder facilitate riboflavin biosynthesis and the yeast extract have negative effect. It was found that the concentrations of inorganic ions and the amino acids in yeast extract were much higher than that of the yeast powder. The inorganic ions and the amino acids were respectively added to the medium while yeast powder was used as base nitrogen source. The fermentation results show that excess inorganic ions and glutamine have negative effect on riboflavin biosynthesis. The yeast extract restrained riboflavin biosynthesis for it contains much glutamic acid.