Abstract: To improve the survival rate of Lactiplantibacillus plantarum during freeze-drying, this study developed a composite cryoprotectant and prepared freeze-dried bacterial powder using vacuum freeze-drying technology. The composition of the composite cryoprotectant was optimized through single-factor and orthogonal experiments to enhance bacterial viability in the freeze-dried powder. The protective effects were evaluated by comparing viability before and after freeze-drying, measuring cell membrane and cell wall integrity, observing cellular microstructure, and assessing storage stability. The protective mechanism was further investigated via Fourier-transform infrared spectroscopy analysis of the freeze-dried powder, differential scanning calorimetry to determine thermal properties, quantification of subcellular damage, Live-dead cell staining, and lactate dehydrogenase activity assays. Results showed that the optimal composite cryoprotectant formulation consisted of 0.15 g/mL skim milk powder, 0.06 g/mL trehalose, 0.03 g/mL Tween 80, and 0.014 g/mL L-glutamic acid. After 60 days of storage at −20 ℃, the freeze-dried powder exhibited a survival rate of 59.64%±1.74% and a viable count of (4.58±0.13)×10⁹ CFU/mL. Scanning electron microscopy revealed that cells treated with the composite cryoprotectant maintained intact morphology with smooth surfaces. Fourier transform infrared spectroscopy analysis indicated integration of cryoprotectant components into bacterial cells, strengthening hydrogen bonding. Differential scanning calorimetry results demonstrated enhanced thermal stability of the freeze-dried powder. Cell membrane and wall damage assays showed significant (P<0.001) reduction in structural damage, while lactate dehydrogenase activity increased significantly (P<0.0001). Correlation analysis suggested that the composite cryoprotectant improved survival primarily by preserving membrane integrity and enhancing lactate dehydrogenase activity. This study developed an effective composite cryoprotectant for Lactiplantibacillus plantarum. It elucidated its protective mechanisms, providing a theoretical basis for the application of cryoprotectants in probiotics, such as lactic acid bacteria.