Objective Artificially formulated milk fed after birth is essential for cultivating germ-free rats. Lactose-free milk powder with deep hydrolyzed protein is easily absorbed by infants and reduces indigestion and allergic reactions. We detected the biochemical indexes of germ-free rats to investigate whether adding lactose-free milk powder to their milk would have a positive effect on the rats. Methods Pregnant Sprague-Dawley rats were determined to be in labor. After surgically removing the uterus, the young rats were housed in isolators and artificially lactated for 22 days. Body weight and survival rates were recorded. The experimental group received lactose-free milk powder with deep hydrolyzed protein in their milk, and the control group received regular formula milk. The blood biochemical indexes were measured at 8 weeks. Results During the first 14 days of artificial feeding, body weight and survival rates did not differ between the experimental and control groups. The weights and survival rates of the experimental group began to increase compared with those of the control group on day 14. The survival rate and body weights of the experimental group were significantly higher than those of the control group on day 22 [ 37. 18% vs 17. 78% and ( 9. 96 ± 0. 49) g vs ( 13. 36 ± 0. 59) g, respectively]. Blood biochemical indexes of the germ-free rats showed that their aspartate aminotransferase levels decreased, and their glucose levels increased. Conclusions In cultivating germ-free rat pups, lactose-free milk powder with deep hydrolyzed protein can be added to formula milk to effectively increase the weight of the young rats and reduce their mortality rate. Adding lactose-free milk powder to formula milk substantially changed the blood biochemical indexes of female germ-free rats.