This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: $99.5{pm}0.8%$, DMSO 7.5%: $99.5{pm}0.7%$, and DMSO 10.0%: $99.6{pm}0.6%$). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: $96.2{pm}2.3%$, DMSO 7.5%: $95.3{pm}3.6%$, 10.0%: $96.6{pm}1.8%$) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm ($98.4%{pm}0.5$) and DMSO concentration level of 5.0% ($97.8{pm}0.1%$) were not significantly different. However, with 7.5% ($97.2{pm}0.6%$) and 10.0% DMSO concentrations ($95.9{pm}0.2%$) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; $66.8{pm}1.8%$, HR: $82.0{pm}12.9%$) and 2 years (FR; $78.5{pm}14.8%$, HR: $79.3{pm}0.6%$) cryopreserved sperm were lower than control (FR; 100%, HR: $91.1{pm}3.6%$) and 2 days cryopreserved sperm (FR; $99.6{pm}0.6%$, HR: $96.6{pm}1.8%$). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.