Cane molasses (CM), an agro-industrial by-product, was first examined for docosahexaenoic acid (DHA) production by Schizochytrium sp. Cell dry weight as 21.94 g/L at treated CM cultivation was similar to that at pure glucose (26.7 g/L) cultivation. Batch fermentation at different initial CM concentration showed that DHA percentage could reach 47.51% at 10 g/L CM but only 37.90% at 70 g/L CM. By analyzing the fermentation process, monosodium glutamate might be a positive agent for effective DHA production. Finally, monosodium glutamate and malic acid were introduced to the fed-batch fermentation for effective DHA production.