Hyaluronidase was produced by submerged fermentation from Streptococcus mitis. The possibility of using Streptococcus mitis for enzyme production has been recently investigated. In this study, the physical and nutritional parameters were optimized to improve the production of hyaluronidase by Streptococcus mitis and it was accessed. Maximum production of hyaluronidase was obtained when 5% starch supplemented as carbon source achieved by employing (98.7U/ml) and followed by ammonium chloride (140.4U/ml) incubation period about 48 hours showed (108.9U/ml) and temperature at 37°C showed (179.9U/ml). The maximum enzyme yield on pH 4 is (110.7U/ml). The production of hyaluronidase by means of immobilized Streptococcus mitis was evaluated and a maximum production was obtained with the medium was inoculated with 100 beads (591U/ml) which was more than that of mobilized cells.