[Objective] In order to optimize the cultivation conditions of the Schizochytrium sp. 20888 to increase DHA yield. [Methods] The effects of cultivation time, carbon and nitrogen sources and their concentrations and culture temperature on the cell growth, cellular lipid and DHA contents were studied. A series of “one-factor-at-a-time” experiments and orthogonally designed tests were carried out. The biomass was tested as dried weight, the lipid content was tested by Soxhlet extraction and the DHA content was determined by gas chromatography. [Results] The optimal cultivation time was 4 d, the proper temperature was 23 °C, and the optimal medium formula was as following (g/L): glucose 65, glycerol 80, peptone 6, yeast extract 4, sodium glutamate 8. [Conclusion] Optimal cultivation conditions for DHA production were obtained successfully. Under the optimal conditions, the DHA yield was reached to 33.68%.