Cassava (Manihot esculenta Crantz), as an important food and economic crop, has significant implications for the sustainable development of breeding work and food security through the preservation of its germplasm resources. Cryopreservation of shoot tips, which saves space and labor, and reduces the risk of genetic variation during storage, is an important method for the long-term preservation of germplasm resources of vegetatively propagated crops like cassava. The droplet-vitrification method is currently a widely used technique for shoot tip cryopreservation, but it has not been optimized for cassava shoot tips, limiting its application for cassava long-term conservation. Therefore, this study used hormone-free MS medium as the recovery medium to optimize key steps in the droplet-vitrification cryopreservation process for cassava shoot tips. The study found that a 4-hour preculture in 0.3 mol/L sucrose MS medium before loading solution treatment, combined with 50-60 minutes of cryoprotection on ice using PVS2 plant vitrification solution, significantly enhanced the cryopreservation effect, resulting in an 85.6% survival rate and a 63.3% regeneration rate for cassava 'COL777' shoot tips after ultra-low temperature preservation. In a genotype applicability test involving 15 cassava genotypes, 8 of them achieved regeneration rates ranging from 33.3% to 73.3% after ultra-low temperature preservation. However, the cryopreservation effects were unsatisfactory for 7 genotypes. Consequently, the study subsequently optimized the regeneration medium following shoot tip cryopreservation. It was discovered that removing auxin (NAA) and adding a lower concentration of 6-benzylaminopurine (6-BA) to the regeneration medium prevented the formation of callus tissue from the cryopreserved shoot tips, thereby improving the regeneration rate. Ultimately, an optimized regeneration medium, RMB2, was developed: 0.09 μmol/L 6-BA + 0.23 μmol/L gibberellic acid (GA3) + 4.4 g/L MS + 30 g/L sucrose + 7 g/L agar. When cassava 'COL777' and 'SC8002' were cultivated on RMB2 after cryopreservation, their regeneration rates were 86% and 69%, respectively. In a test involving 7 genotypes using RMB2, 4 of them achieved shoot tip regeneration rates above 30% after cryopreservation. Overall, 80% of the cassava genotypes tested achieved regeneration rates above 30% after cryopreservation using the optimized ultra-low temperature preservation technique. This result also enhances the genotype applicability of the droplet-vitrification method. Therefore, the droplet-vitrification ultra-low temperature preservation system established in this study is simple and efficient, significantly promoting the development of cassava germplasm cryopreservation work and better protecting cassava germplasm resources.