The influence of salinity, carbon and nitrogen sources of growth media on the production of microbial docosahexaenoic acid (DHA) by Schizochytrium sp. was investigated. The obtained results indicated that the strain Schhochytrium sp. showed a wide tolerance to salinity, that is, the optimum salinity was between 50 and 200% in respect to the salt concentration of seawater. Glucose and fructose were the most suitable carbon sources for cell growth as well as DHA accumulation. The mixed-organic nitrogen source led to more DHA yield than the single nitrogen source. Box-Behnken design and surface response methodology were employed to formulate the optimal medium. The optimized medium contained 126.20 g-L-1 glucose, 10.06 g-L-1 yeast extract, 2.06 g-L-1 corn steep liquor and 5.00 g-L-1 soy peptone in seawater at half salt concentration. The maximum biomass, total fatty acid and DHA yield reached 42.9, 34.1 and 13.8 g-L-1 on the optimized culture medium after 5 d of cultivation, respectively. Thus Schizochytrium sp. was proved to be a promising resource for microbial DHA production owing to its high level of productivity after medium optimization.