This research was to determine the optimum concentration of egg yolk on spermatozoa motility of Giant Grouper 48 hours after cryopreservation. Egg yolk used in this study were 0 %, 5 %, 10 %, 15 %, 20 % and 25 %, respectively, combined with 6 % glycerol. Sperm was collected by a hand stripping method. Sperms were then conditioned at 4 °C for 10 minutes and were refrigerated at -20 °C for 48 hours. Thawing was carried out at 4 °C for 30-60 seconds. Spermatozoa motility was evaluated before and after frozen. According to Anova test, there was a notable impact (p≤0.05) from a varied contents of egg yolk on post-thaw spermatozoa motility. Based on the Tukey test, the concentration of 15 % of egg yolk showed the highest post-thaw spermatozoa motility (83.64±1.72 %) on post-thaw motility (p<0.05). Fifteen percent of egg yolk demonstrated the optimum concentration in this study.