Abstract: In order to investigate the effects of chemical modulators on the synthesis of fatty acids ( SFAs, PUFAs and DHA) in Schizochytrium limacinum mutant strain, the composition of fatty acids in Schizochytrium limacinum mutant strain was analyzed by chromatography-mass spectrometry ( GC-MS) .The optimum addition conditions of three chemical modulators, ethanolamine ( ETA) , naphthalene acetic acid ( BNOA) and salicylic acid ( SA) were optimized by single factor and orthogonal design.The results showed that the adding ethanol amine 150 mg/L ( ETA) , 10 mg/L naphthoxyacetic acid ( BNOA) and 1 mg/L salicylic acid ( SA) , the DHA production of Schizochytrium limacinum mutant strain was the highest, reaching 6.18 g/L, which was12.77% higher than control group.It was suggested that the adding appropriate amount of chemical modulators could improve DHA content of Schizochytrium limacinum mutant strain.