Abstract: This study was aimed to explore the effects of different methyl esterification methods on the production of fatty acids from Schizochytrium. In this paper, the pure algae bodies were obtained by fermentation and culture, then the dried algae were acquired by enzymatic breaking and freeze-drying, soxhlet extraction method was used to extract the oil from the dried mycelium of Schizochytrium. The fatty acids in algal oil were esterified by three different methyl esterification methods, the composition and content of DHA(Docosahexaenoic acid)fatty acids in algal oil were analyzed by gas chromatography-mass spectrometry(GC-MS)technique. The results showed that 11 fatty acids were identified by acid-catalyzed esterification method, 19 fatty acids were identified by alkali-catalyzed esterification method and 12 fatty acids were identified by BF₃-catalyzed esterification method. The contents of the identified fatty acids accounted for 89.50%, 97.88% and 90.12% of oil of Schizochytrium, respectively. The content of DHA accounted for 38.89%, 46.76% and 40.54% of the total fatty acids, respectively. Therefore, alkali-catalyzed esterification method compared to acid-catalyzed and BF₃-catalyzed esterification method had obvious advantages, more suitable for fatty acid analysis of algae oil in Schizochytrium. Further more, the esterification temperature of the optimal alkali-catalyzed esterification method was conducted a gradient experiment, and the result showed that 55℃ was the optimal esterification temperature and the relative content of DHA was 47.45%. In a word, this research had an important significance for the detection of polyunsaturated fatty acid with long chain.