Abstract: Corn flour as a main raw material for producing L-lactic acid, not only be able to improve corn's values in use, but also be able to reduce the production cost of L-lactic acid. The corn sacchrification liquid by double-enzyme hydrolysis could be a main carbon source for L-lactic aid fermentation, then suitable organic nitrogen sources for lactic acid bacteria TL-2 was explored. Results show that peptide-3 can be organic nitrogen in the fermentation culture medium to produce L-lactic acid through comparing the TL-2 growth condition in several different flat media. When organic nitrogen sources and their concentrations are changed on the basis of initial medium, the same yield of L-lactic acid is obtained, but the color of fermentation liquid which containing 10 g/L peptide-3 is the lightest. Response surface methodology were applied to optimize three factors of medium. When the ratio of sugar concentration to peptide-3 is 15.5∶1, shaking time is 24.27 h, the yield of L-lactic acid is the largest. The fermentation form and optimized medium allow lactic acid yield to 143.8g/L in flask-shaking, sugar utilization to 95.8%, fermentation period shortens to 60 h.