Cryopreservation of fish sperm is a new technology in fisheries science and still need to be developed for the sake of fish biodiversity and conservation. Nowadays, many fish species are critically endangered or vulnerable due to over exploitation, habitat degradation and so on. Rohu fish is one of the most consumed fish species in south-east Asia, especially in Bangladesh. The aim of the study is to develop and standardize a suitable cryopreservation protocol for Rohu fish sperm and therefore, three experiments were conducted. Firstly, to determine the effect of storage period on post-thaw motility of sperm. Secondly, to determine the optimal egg : sperm ratio for sustainable use of cryopreserved sperm. Finally, to determine the effect of cryopreserved sperm on fertilization and hatching.