Abstract: In this study, the utilization of 16 nitrogen sources of Lactobacillus delbrueckii subsp. bulgaricus B61-3 were determined, and the types and amounts of nitrogen sources that can improve the freeze-drying tolerance of the strain were screened out. More importantly, the effects of nitrogen source modification on the fermentation performance, cell morphology and enzyme activities of freeze-dried powder of the strain were investigated. The results showed that 30 g/L of bovine bone peptone improved the freeze-drying survival rate from 9.68% to 18.90%, and increased fermentation activity by 22.15% compared with the control group. Changed cell size and morphology of the cultured cells of bovine bone peptone were also observed by electron microscope. Cell cultured in bovine bone peptone medium represented a short rod with smooth surface, and the ratio of length to diameter or area to volume decreased significantly (P<0.05). However, cells in the control group showed irregular, curly shape and longer cells. Freeze-drying significantly reduced the enzyme activity of the strain (P<0.05). Compared with control group, the intracellular activities of these enzymes in the cells cultured in bovine bone peptone significantly increased after freeze-drying (P<0.05), and the activities of lactate dehydrogenase, β-galactosidase and Na⁺K⁺-ATPase increased by 1.30 times, 1.52 times and 2.75 times respectively, while the extracellular activities of β-galactosidase decreased. Meanwhile, the results showed that B61-3 cells cultured with bovine bone peptone could resist the damage of cell membrane caused by freeze-drying process, reduce the leakage of enzymes, thus improving the freeze-drying survival rate and fermentation activity.