The Chinese sturgeon (Acipenser sinensis) is one of the key aquatic animals under first-degree protection in China, but its germplasm resources, such as spermatogonial stem cells, are not well protected by cryoprotection technology. In this study, we investigated the effects of thawing temperature, antioxidants, and antifreeze proteins on the cryopreservation of spermatogonial stem cells of Chinese sturgeon based on the formulation of the spermatogonial stem cell cryopreservation solution used for the American paddlefish (Polyodon spathula). The aim was to establish a highly efficient method for ultra-low-temperature cryopreservation of spermatogonial stem cells of Chinese sturgeon. The best cryopreservation effect was achieved at a thawing temperature of 25 ℃ among the temperatures tested (20, 25, 30, and 40 ℃). The number of cells after thawing was (3.86±0.51)×10⁵ , and the cell viability reached (96.36±0.53)%. The effects of different concentrations (500 mg/L, 1000 mg/L, and 1500 mg/L) of glutathione, ascorbic acid, and α-tocopherol on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined. The results showed that the experimental group with the addition of 1000 mg/L α-tocopherol to the cryopreservation solution had the highest number of cells (7.64±0.34)×10⁵ after thawing, which was significantly higher than that of the other groups with antioxidants added, and the cell viability reached (92.82±0.72)%. The effects of different concentrations (0.1, 1.0, and 10 μg/mL) of two types of antifreeze proteins (AFPI and AFPIII) on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined. The experimental group with 1.0 μg/mL AFPI had the best defrosting effect, and the number of cells obtained after defrosting was (6.85±0.19)×10⁵ , which was significantly higher than the number in the other experimental groups, and the cell viability was (86.89±0.73)%. In summary, in the present study, we obtained the optimal thawing temperature for the cryopreservation of spermatogonial stem cells of Chinese sturgeon, evaluated the effects of three types of antioxidants and antifreeze proteins on the effect of cryopreservation, and established a highly efficient cryopreservation technology for spermatogonial stem cells of Chinese sturgeon. These data provide technical support for the cryopreservation and restoration of the Chinese sturgeon’s germplasm resources.