〔Abstract〕 Objective To explore the predictive value of combined detection of serum estradiol (E2), progesterone (P), and β-human chorionic gonadotropin (β-HCG) in the second trimester of twin pregnancy for maternal and fetal outcomes. Methods A total of 60 twin pregnancies in the second trimester who underwent regular prenatal examination at Ruzhou Maternal and Child Health Hospital from June 2018 to March 2021 were included in the twin group. Additionally, 98 singleton pregnancies in the second trimester who underwent prenatal examination at the same hospital during the same period were included in the singleton group. The levels of serum E2, P, and β-HCG in the second trimester were compared between the two groups. According to the delivery outcomes, the twin group was divided into normal subgroups, singleton adverse subgroups, and twin adverse subgroups. The correlation between the levels of serum E2, P, and β-HCG in the second trimester and maternal and fetal outcomes in the twin group was analyzed. Results The levels of serum β-HCG, E2, and P in the second trimester of the twin group were higher than those in the singleton group, with statistically significant differences (P < 0.05). The levels of serum β-HCG, E2, and P in the singleton adverse subgroups and twin adverse subgroups were lower than those in the normal subgroups, and the levels in the twin adverse subgroups were lower than those in the singleton adverse subgroups, with statistically significant differences (P < 0.05). There was a high negative correlation between maternal and fetal adverse outcomes and the levels of serum β-HCG, E2, and P in the second trimester, with statistically significant differences (P < 0.05). Conclusion The levels of serum E2, P, and β-HCG in the second trimester of twin pregnancy are higher than those in singleton pregnancy. The risk of adverse outcomes in twin pregnancy increases with the abnormal reduction of serum E2, P, and β-HCG levels.