For establishing an efficient and stable mutagenesis method on breeding Schizochytrium DHA higher producers, the wild-type strain named Schizochytrium sp. 31 was mutated combining Ethylmethylsulfone (EMS) and Atmospheric and Room Temperature Plasma (ARTP) treatment. Mutagenic efficiency, fermentation indexes of the mutants were compared and analyzed. Under this compound mutagenesis method, the positive mutation rate reached 32.2%, which was much higher than that under other two single-factor mutagenesis methods. Besides, DHA production and DHA percentage in total fatty acids reached to 7.2 g/L and 43.2%, which were 35.6% and 19.2% higher than that of the wild-type strain, respectively. After five rounds of cell culture passages, mutants induced from compound mutagenesis method showed up with better genetic stability. This mutagenesis method was proved to be an efficient strategy of breeding Schizochytrium DHA higher producers. It also provideed references to the breeding practices on the algal higher producers for synthesizing other types of polyunsaturated fatty acids.