In order to investigate the position of various fatty acids attached to glycerol and the specificity of Lipolase-100T, hydrolysis of fish oil was carried out with Lipolase-100T derived from Aspergillus oryzae. The amounts of free fatty acids produced from triglyceride, 1,2(2,3)-diglyceride, 1,3-diglyceride, and 2-monoglyceride and conversion rates of 1,2(2,3)-diglyceride to 1,3-diglyceride and 2-monoglyceride to 1(3)-monoglyceride were also calculated. The ratio of 1,2-diglyceride content to 1,3-diglyceride was higher than 70 in the early period of hydrolysis. The fatty acid content of the glyceride mixture after 72 h of hydrolysis was compared with that of fish oil, and it was found that polyunsaturated fatty acids such as C16:4, C20:4 n-3, C20:5 n-3, C21:5 n-3, C22:5 n-3 and C22:6 n-3 were located in the 2-position of glycerol. Material balance of each component in the hydrolysis system was written to obtain a set of simultaneous linear equations. The theoretical quantity of free fatty acids produced from triglyceride, 1,2-diglyceride, 1,3-diglyceride, and monoglyceride, respectively, were calculated by solving the linear equation system. The conversion rate of 1,2(2,3)-diglyceride to 1,3-diglyceride and that of 2-monoglyceride to 1(3)-monoglyceride were also obtained. The results showed that the migration rate of 1,2(2,3)-diglyceride to 1,3-diglyceride was higher than the hydrolysis rate of 1,2(2,3)-diglyceride to 2-monoglyceride and the conversion rate of 2-monoglyceride to 1(3)-monoglyceride was extremely low.