Eicosapentaenoic acid （EPA） is a kind of n3-polyunsaturated fatty acid with important physiological functions.It has broad commercial prospects in health food，medicine，feed and other industries.However，the deep-sea fish oil as the traditional source of EPA cannot meet the increasing market demand.In this paper，Schizochytrium was taken as the original strain，and a two-stage temperature and low-dissolved-oxygen（TST-LDO） strategy is proposed.The effect of fermentation temperature was optimized by single factor experiment at the shaking flask level，and the effects of two-stage temperature change time and dissolved oxygen on the fermentation performance of EPA production in Schizochytrium were studied in a 5L fermenter.The change of fatty acid components in the process of fermentation was studied.The results show that the highest EPA content （8.35%） is obtained at two-stage-temperature strategy （28℃（0~24h）→34℃（24~120h））.The optimal temperature change time is 24h，which is determined in a 5 L fermenter.The results of different dissolved oxygen studies show that the medium and high dissolved oxygen （10%DO and 25%DO） are more conducive to the growth and lipid accumulation of Schizochytrium，but the low dissolved oxygen （2%DO） is more conducive to the accumulation of EPA，and the content of EPA is 2.05 times and 4.98 times of the medium and high dissolved oxygen level，respectively.When fermentation reached 96 h under TST-LDO strategy，the biomass，lipid yield，EPA content in total fatty acid and EPA yield reached 56.60g/L，9.62g/L，12.50% and 1.14g/L，respectively.Compared with the pretreatment，EPA content and EPA yield increased by 14.5 times and 6.1 times，respectively.