Longan (Dimocarpus longan Lour.) is native to southern China and widely cultivated in the southern and southeastern regions. It is a fruit tree with high economic value. Longan seeds, as a typical recalcitrant seed, are highly sensitive to low temperature and drying, making them difficult to preserve in traditional low-temperature seed banks. After excluding the influence of ice crystals on seeds, this study conducted vitrification freezing and direct freezing ultra-low temperature storage on fresh and dry seeds, and compared the changes in physiological and biochemical indicators, contents, and starch. After dehydration and ultra-low temperature storage, longan seeds almost lost the vitality. Compared with healthy seeds, dehydration led to a significant increase in malondialdehyde content and severe damage to cell membranes. The hydrogen peroxide content showed a decreasing trend. The expression of antioxidant enzyme system increased, and the highest SOD activity was observed in longan seeds with a water content of 16% after vitrification and freezing treatment, while vitrification and freezing treatment reduced the activities of POD and CAT. Low temperature and dehydration led to an increase in the content of soluble sugars and proteins in seeds, while dehydration led to protein denaturation and a decrease in content. Observing the starch of longan seeds, it was found that after vitrification and freezing treatment, there were voids and depressions on the surface of starch particles, which were directly frozen and caused starch particle rupture. Dehydration and low temperature did not affect the molecular structure of starch, but caused some degree of damage to the internal ordered structure.