The heterotrophic microalgae Crypthecodinium cohnii was usually cultivated in complex medium containing glucose, yeast extract and sea salt. For the preparation of DHA with highest purity, a new defined medium without the yeast extract was developed. Different inoculated densities, C/N ratios, temperatures, culture volumes and glucose additions were investigated to optimize the algal growth rate and DHA production. The growth period in C. cohnii was shortened from 12-14 days to 7-8 days, the OD600 was enhanced from 2.0 to 3.0, the glucose consumption was accelerated and used up on day 3-4, and the DHA content in culture were increased from 10 to 45 nmoles/300 μl batch. It was found that C. cohnii had optimal growth and DHA accumulation in 25 °C, 0.2 inoculated density, 5-10 C/N ratio, 5:1 air/culture volume ratio. This is the first time DHA production using C.cohnii has been optimized in synthetic medium. This allows preparation of uniformly radiolabeled 13C- and 14C-DHA.