The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.