Antioxidant peptide 3-2 which is isolated and purified in whey protein hydrolysate for the protection of Bifidobacterium animalis subsp. lactis Probio-M8 (B. lactis Probio-M8) during vacuum freeze-drying. Whey protein was added to a basal lyoprotectant containing 10% trehalose as a control group and antioxidant peptide 3-2 was added as an experimental group. After freeze-drying， the viable count， cell membrane structure， intracellular and extracellular pH， as well as intracellular ROS levels and malondialdehyde content of lipid oxidation products due to oxidative stress， were measured in Probio-M8. Results: Antioxidant peptide HP3-2 (5 mg/mL) could improve the survival rate (80.40 ± 3.34)% of Probio-M8 after freeze-drying， which was significantly higher than the control group of whey protein (64.69 ± 2.47)%. It was also able to reduce the intracellular reactive oxygen species， which resulted in the content of malondialdehyde (2.14 ± 0.06) nmol/mL， a product of lipid oxidation， to be significantly lower than WP group (2.30 ± 0.05) nmol/mL and T group (2.33 ± 0.05) nmol/mL. It also could maintain the cell membrane potential and proton gradient， and the unsaturated fatty acid of the cell membrane (51.49 ± 1.26) was significantly higher than WP group (39.96 ± 1.57) and T group (33.33 ± 0.62)， thus increasing cell membrane fluidity. Conclusion: Antioxidant peptide 3-2 can protect the Probio-M8 during freeze-drying.