Fish semen cryopreservation is a valuable technique to sustain good quality sperm for fertilization, facilitating threatened species conservation and broodstock genetic development. The present investigation aimed to develop suitable standardized protocols to cryopreserve sea bass (Dicentrarchus labrax) semen. Thus, four experiments were conducted to determine the most effective extender (egg-yolk citrate, urea-egg-yolk, and 0.9% NaCl), cryoprotectant (DMSO, glycerol, ethylene glycol “EG", and methanol), sperm/cryodiluent dilution ratio (1:2, 1:4, 1:10, and 1:20) and equilibration time (0, 5, 15, or 30 min) to maintain the sperm content after cryopreservation. The consistency of post-thaw semen was assessed, with motility percentage, motility duration, and fertilization rate all being taken into account. When sperms were thawed, the consistency was at its best, especially when sperm was diluted with a ratio of 1:20 with egg-yolk citrate extender containing 10% DMSO, with a 15-min equilibration period before cryopreservation. Sea bass sperm cryopreserved using this protocol exhibited fertilisation rates, class motility, and motility duration close to fresh semen. Thus, the cryopreservation method developed in the present investigation proved to be an efficient and effective approach to augment the cryosurvival of sea bass spermatozoa.