Abstract: Monofactorial experiments were performed to study the effects of six operational parameters,including fermentation means, fermentation duration, loading capacity, inoculum size, temperature and initial p H, on the preparation of L-lactic acid by the fermentation of waste tobacco stems with Lactobacillus casei XJL. According to the results of monofactorial experiments, four factors(inoculum size, loading capacity, initial p H and temperature) were selected and orthogonally tested at three levels.The results showed that: 1)Initial p H, inoculum size and loading capacity significantly affected the yield of L-lactic acid. 2)The optimum operational parameters were initial p H 7.0, loading capacity 120/250 m L(120 m L tobacco stem extraction loaded in a conical flask of 250 m L), inoculum size 7.5%(the volume ratio of transferred inoculant solution to total culture solution after inoculation), static culture for 48 hours at 37 ℃; under the above conditions, the maximum yield of L-lactic acid reached 157.50 g/kg.