Abstract: On the basis of screening the optimal concentration, the content of gibberellin, auxin and abscisic acid in different layering stages were determined by LC-MS. The dynamic changes of three hormones and their interactions in seed dormancy were analyzed, and the mechanism of endogenous hormones in dormancy release of Cardiocrinum giganteum seeds was explored. Results show that the length of C. giganteum embryo increases with the increase of the stratification time. When the layer is 135 d, the embryo length of 5 mg/L 2,4-D is 9.03% higher than CK. Under 5 mg/L 2,4-D treatment, the growth rate of the embryo is the highest, reaching 82%, and the germination initiation time is the earliest, after 14 days at 4℃; the germination rate is the highest, reaching 75.33%. In the process of stratification, the changes of abscisic acid content in CK and 2,4-D treatments show a general downward trend, and both auxin and gibberellin content show a trend of increasing first and then decreasing. The auxin content peaks at 60 d, CK is 516.66 ng/g higher than 2,4-D treatment, gibberellin peaks at 120 d, and 2,4-D is 52.17 ng/g higher than CK. The ratio of auxin to abscisic acid, gibberellin to abscisic acid increase first and then decrease. When the layer is 60 d, the ratio of auxin to abscisic acid peaks, and CK is 49.24 times higher than 2,4-D. When the layer is 120 d, the ratio of auxin to abscisic acid treats by 2,4-D is 10.53 times higher than that of CK. When the layer is 120 d, the ratio of gibberellin to abscisic acid peaks, and 2,4-D treatment is 1.63 times higher than CK. It can be seen that 5 mg/L 2,4-D treatment is the best concentration, which is beneficial to seed dormancy release and germination. The change of abscisic acid content is negatively correlated with the development of C. giganteum embryo. The auxin and gibberellin may be the main regulators to promote the dormancy release of C. giganteum seed.