A method was established for the simultaneous determination of 3 kinds of characteristic markers in DHA soft capsules from different sources. The other physicochemical indexes were comprehensive assessed. The method was: after methyl esterification and derivatization, the methyl esters of DHA, EPA and linoleic acid in different DHA soft capsules were detected by external standard method, and then the contents of the corresponding fatty acid were obtained by conversion. The product characteristics, acid value, peroxide value and arsenic of 4 samples from different companies were in line with relevant national standards. The results of verification test for the determination of DHA methyl ester, EPA methyl ester and linoleic acid methyl ester were as follows: the precisions were 1.39%, 1.43%, 0.5%; the linear ranges were 0.3～2.4mg/mL, 0.2～2.4mg/mL, 0.1～1.6mg/mL; the correlation coefficients were 1.000, 0.9999 and 1.000, which showed good linear relationship; the average recovery ranges were 96.9%～101.4%, 97.5%～100.3%, 95.4%～99.6% ,respectively, which showed that the method had high accuracy; the concentrations of the limit of quantitation (LOQ) were 0.08, 0.04, 0.03mg/mL, respectively. The stability data (RSD) within 24h were 3.19%, 3.65%, 3.02%, which indicated that the sample had good stability.The evaluation results of DHA soft capsule showed that the 3# had high acid value and slow disintegration, the 2# had low content of DHA and linoleic acid, and the 0# had the highest content of linoleic acid among the samples. Compared with other physicochemical indicators, the difference among 4 brand products on the 3 kinds of characteristic markers were larger and the markers were suitable to reflect the key quality.