Endogenous estrogen and progesterone and their metabolites play a key role in regulating and maintaining the normal pregnancy process. However, the dynamic change of these estrogen and progesterone metabolites' level across the entire gestational period is not fully revealed. This study systematically measures the temporal changes of estrogen, progesterone, and their metabolites in human urine during normal pregnancy using ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). We find that the levels of estrone, estradiol, estriol, 16-epiestriol, 17-epiestriol, 2-methoxyestradiol, and 4-hydroxyestrone gradually increase during pregnancy. The levels of 2-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol rapidly decrease in the early pregnancy and then maintain at lower levels. The levels of 4-methoxyestradiol, 4-methoxyestrone and 2-methoxyestrone peak in the mid-pregnancy and then gradually decrease. The levels of pregnenolone, 17α-hydroxy pregnenolone, 17α-hydroxy progesterone, pregnanolone, epipregnanolone gradually increase during pregnancy. The levels of progesterone, 20α-hydroxy progesterone, 5α-dihydroprogesterone and 5β-dihydroprogesterone first increase in the mid pregnancy and then decrease in the late pregnancy. In sum, this study comprehensively depicts the dynamic change of estrogen, progesterone, and their metabolites in human urine during pregnancy, which lays the foundation for hormone monitoring and early diagnosis of pregnancy complications, and further mechanistic study of the roles of these metabolites in pregnancy.