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There is significant demand for high-purity DHA in the pharmaceutical industry. Traditionally, this high-purity DHA is extracted from raw materials with relatively low DHA content (10–20%), such as fish oil. Recently, through electroporation-induced mutation, a high-DHA-content strain of <i>Schizochytrium</i> sp. GCD2032 was isolated. To further enhance its DHA production, optimizations were conducted on the culture medium and fermentation conditions (in shaking flasks), as well as different nitrogen source concentrations (in a 5 L fermenter) for biomass, fatty acid content, and DHA content (as a percentage of total fatty acids). In a 5 L fermenter, <i>Schizochytrium</i> sp. GCD2032 achieved a biomass of 50 g/L, with fatty acid content of 55.71% and DHA content of 61.29%. Notably, the DHA content reached an impressive 341.45 mg/g of dry weight. This strain consistently produces high levels of fatty acids and DHA, demonstrating significant potential for pharmaceutical applications.