The culture conditions for the production of carrageenase were optimized using one-factor-at-a-time method combined with orthogonal array design. With the one-factor-at-a-time method revealed optimal conditions for carrageenase production were 24 h of fermentation period, 28 °C incubation temperature at pH 8.0 with NaNO3 as nitrogen source and carrageenan as carbon source in MMS media. Further optimization of carrgeenase production by using orthogonal experimental design L9 (34) with four factors, temperature, pH, NH4NO3 and carrageenan with their relevant levels revealed optimised conditions for carrageenase production were temperature of 28 °C, pH 8.0, 2 g L-1 NaNO3 and 2 g L-1 carrageenan. The order of the factors affecting the fermentation process was found to be temperature > pH > NaNO3 > carrageenan. The temperature played a significant role on the carrageenase production. Higher carrageenase yield with activity of 0.542 ±0.045 U ml-1 was obtained in the optimised medium when compared to those of basal medium. Carrageenase hydrolysed products of carrageenan were identified by LC-ESI-MS as neocarrabiose, neocarrabiose-4 sulfate, neocarratetraose, neocarratetraose-4 sulfate, anhydrogalactose, galactose, galactose-4 sulphate and sulphate.