In this paper, we studied the spermatozoa cryopreservation of Nibea albiflora using two-step cooling methods, HBSS as extender, EG(ethylene glycol) as cryoprotectant, in 0.5mL straws, also investigated the DNA damage in response to the cryopreservation process by SCGE. The results showed that there were no significant differences between fresh sperm and frozen-thawed sperm which diluted with 5%, 10% or 15%EG in moving time and life-span time. However, frozen-thawed sperm moving time and life-span time with 20%, 25% or 30% EG as cryoprotectant had a significant drop as compared with fresh sperm. The SCGE results demonstrated that there were no significant differences between fresh sperm and frozen-thawed sperm which diluted with 5%, 10%, 15% or 20%EG in DNA fragments, but DNA fragments differed significantly with fresh sperm when EG concentrations was added to 25% or 30%. In fact, there was a positive correlation between DNA damage extent of frozen-thawed sperm and concentrations of EG in protocol. So we conclude that cryoprotectant of 5%~15%EG can be considered as the most effective choices for spermatozoa cryopreservation of Nibea albiflora.