Objective： The effects of different culture conditions on expression of key extracellular gene PrtB of the protein hydrolysis system of Lactobacillus bulgarian was evaluated in the present experiment. Methods： Real-time PCR technology was used to determine target gene expression among different strains under different growth periods and different nitrogen sources， initial pH（5.6，5.9，6.2，6.5） and temperature（30，37，42，45 ℃）. The experimental results showed that the expression levels of PrtB were significantly reduced in three strains of Lactobacillus bulgaricus LB08012, LB08014 and LB08016 from the early logarithm phase to the stable phase（P<0.05）. Under the condition of N lack of culture， the expression of PrtB was significantly higher（P<0.05） than that of normal state culture. In addition， the expression of PrtB was significantly decreased （P<0.05） under the cultivation condition of adding the casein. The expression quantities of PrtB in the medium of pH5.9, 6.2 and 6.5 were significantly increased （P<0.05） in strains LB08006, LB08007, LB08012 and strain LB08016， however， the expression quantities of PrtB in strain LB08014， LB08015 and strain LB08017 was significantly decreased（P<0.05） compared to the control group （pH 5.6）. In liquid MRS medium at different temperatures， the expression of prtb of strains LB 08007, LB 08012, LB 08014 and LB 08015 increased first and then decreased at 37 ℃, 42 ℃ and 45 ℃， while the prtb expression of strains LB 08006， LB 08016 and LB 08017 increased first. After the reduction. At a temperature of 45 ℃, the expression level of PrtB tended to be 0， which was 10.7 times lower than that of the control group （30 ℃）.