This paper aims to explore the changes in plasma membrane, vitality, ultrastructure and enzyme activity of giant grouper sperm in cryopreservation, and provides a theoretical basis for elucidating the relevant mechanisms affecting the cryopreservation quality of sperm. The semen of giant grouper was collected and stored for 0, 23, 49 and 61 months. Plasma membrane integrity was assessed in fresh sperm and cryopreserved sperm by Y-aniline black staining. The motion parameters of giant grouper sperm before and after cryopreservation were analyzed using computer assisted sperm analysis system. Changes in the activity of six enzymes (SDH, CAT, GR, T-SOD, GSH-Px and CK) ATP content were measured for seminal plasma and sperm. The ultrastructure of fresh and cryopreserved sperm was observed by scanning electron microscope and transmission electron microscope. Fresh sperm had the highest plasma membrane integrity of 83.43%±2.73%, and sperm plasma membrane integrity was significantly reduced after cryopreservation (P<0.05). CASA results showed that the highest mobility was 90.47%±3.34%. Sperm motility was significantly reduced after cryopreservation (P<0.05), but its value stayed within the range of 63.95%±3.66%-68.58%±2.73% after 23-61 months of storage. The VSL VCL and VAP value of cryopreserved sperm had no significant difference compared to fresh sperm (P>0.05). The ultrastructure of fresh sperm showed that the sperm morphology and structure were normal, the mitochondria were arranged regularly, with normal morphology and size. After cryopreservation, the sperm morphology and structural damage was obvious. The damage is manifested as sperm head plasma membrane damage, cytoplasmic leakage, cell nuclear membrane damage, flagella fracture or shedding of the tail. The results of changes in the activities of six enzymes and ATP contents of seminal plasma and spermatozoa before and after cryopreservation freezing of giant grouper sperm showed that after cryopreservation, three enzymes, SOD, GSH-Px and CAT, and ATP contents in spermatozoa were significantly reduced. The enzyme activities in seminal plasma were increased, and all the enzyme activities were significantly different except for GR and CAT. Cryopreservation had a great effect on the enzyme’s activities, ultrastructure and spermatozoa energy of fish. The results have accumulated rich data for the mechanism of frozen fish sperm damage, and provided a technical reference and evaluation index for the long-term cryopreservation of fish sperm.