Abstract: The optimization of fermentation medium for a strain of Lactobacillus R8, which had been proved to be resistant to acid and bile, lays a foundation for the probiotics to be used to improve the gastrointestinal tract healthy food. The components of fermentation medium were optimized by single factor test and orthogonal design test based on the density of bacteria. The results showed that the best fermentation formula for the bacterium was:glucose 20.0 g, yeast powder 30.0 g, MnSO₄·4H₂O 0.075 g, MgSO₄·7H₂O 2.0 g, KH₂PO₄ 2.5 g, citric acid ammonium 2.5 g, CH₃COONa·3H₂O 6.25 g, Tween80 1.0 mL, pH6.2.After optimization, the concentration of bacteria (OD_{600 nm})reached 2.38.