It’s difficult for accurate quantification of astaxanthin esters because of their multiple forms, and hydrolysis of astaxanthin esters to free astaxanthin is the key to solve the problem. In this work, Antarctic krill (Euphausia superba) was used as a representative specie for the study of astaxanthins. The mono- and di-esters of astaxanthin were prepared from Antarctic krill by column chromatography, and the composition and content of them were clarified using high-resolution mass spectrometry with ultraviolet detection. The astaxanthin monoesters and the astaxanthin diesters were then taken as the typical research samples, the enzymatic hydrolysis conditions of which were optimized through single factor and orthogonal experiments, and the accuracy and applicability of the method were evaluated. Results showed that: (1) 8 astaxanthin monoesters and 13 astaxanthin diesters were prepared and identified as typical characteristic compounds from krill. (2) For the astaxanthin monoesters, as the substrate concentration was 0.5 μg/mL, the enzyme concentration of the reaction system was 1.14 U/mL, the reaction temperature was 25 ℃, and the reaction time was 75 mins, the yield of free astaxanthin could reach (94.56±1.24)%; And for the astaxanthin diesters, as the substrate concentration was 1.0 μg/mL, the enzyme concentration of the reaction system was 0.92 U/mL, the reaction temperature was 25 ℃, and the reaction time was 75 minutes, the free astaxanthin yield reached (98.28±0.84)%. (3) Enzymatic hydrolysis at room temperature was applied to the analysis of actual samples, the astaxanthin content was (265.09±20.35) mg/kg in Antarctic krill oil, and the content was (21759.36±90.19) mg/kg in Haematococcus pluvialis. In order to verify the accuracy, the enzymatic hydrolysis method was compared with the standard methods from SC/T 3053—2019 and GB/T 31520—2015. The determination results were 260.42±11.57 mg/kg and 21752.54±100.00 mg/kg respectively, and the deviations of the results were all less than 10%. Then the results were further verified by adding a standard solution of all-trans astaxanthin to the sample matrix was tested, the recovery rate of free astaxanthin was 95.24% in Antarctic krill and 98.56% in Haematococcus pluvialis, RSD was 2.03% and 0.75%, respectively. It proved that the enzymatic hydrolysis method has good accuracy and precision. Studies have shown that the enzymatic hydrolysis method has good stability and high repeatability, reduces the oxidation of astaxanthin in the reaction process, and maximizes the conversion of ester astaxanthin into free form, which is suitable for accurate quantification of esterified astaxanthin. Therefore, the scientific basis for the comprehensive utilization of astaxanthin resources could be provided from this study.