Objective To investigate the predictive value of cervical length (CL) measured by transvaginal ultrasound for preterm birth<32 weeks,＜34 weeks in twin pregnancies in the second and the third trimester of pregnancy. Methods A total of 490 twin pregnant women with CL measured by transvaginal ultrasound during the second trimester of pregnancy (20-24 weeks) and the third trimester of pregnancy (28-32 weeks) delivered in Peking University Third Hospital, and Tongzhou Maternal and Child Health Hospital from January 2014 to December 2017 were collected, and 161 cases out of which were measured by CL during both the second trimester and the third trimester of pregnancy. Based on the measured gestational weeks, 427 cases were in the second trimester group and 224 cases in the third trimester group. The predictive value of CL for preterm birth was evaluated by calculating the optimal cut-off point with sensitivity and specificity. Logistic regression analysis was used to assess the relationship between CL and preterm birth after adjusting for confounding factors (age of pregnant women, chorionic status, mulipara, assisted reproductive pregnancy and pre-pregnancy body mass index). Results (1) The median CL of pregnant women in the second trimester group and the third trimester group were 36 mm (33-40 mm) and 28 mm (18-33 mm) respectively. In the second trimester group, 151 cases (35.4%, 151/427) were preterm birth and 276 cases (64.6%, 276/427) were full-term birth; the median CL of preterm and full-term pregnant women were 34 mm (30-37 mm) and 37 mm (34-40 mm), respectively, with significant difference (P<0.01). In the third trimester group, 100 cases (44.6%, 100/224) were preterm birth and 124 cases (55.4%, 124/224) were full-term birth; the median CL of preterm and full-term pregnant women were 22 mm (15-30 mm) and 31 mm (23-34 mm), respectively, with significant difference (P<0.01). (2) Prediction of preterm birth<32 weeks and<34 weeks was performed with CL in the second trimester group. The area under the receiver-operating characteristics curve were 0.78 (95% CI : 0.70-0.86) and 0.71 (95% CI : 0.64-0.79), respectively. The optimal cut-off points were 36.5 mm and 33.5 mm, respectively. After adjusting for confounding factors, CL was inversely associated with preterm birth<32 weeks and<34 weeks of gestation. (3) Prediction of preterm birth <32 weeks and <34 weeks were performed with CL in the third trimester group. The area under the receiver-operating characteristics curve were 0.86 (0.75-0.96) and 0.75 (0.67-0.84), respectively. The optimal cut-off points were 17.5 mm and 18.5 mm, respectively. After adjusting for confounding factors, CL was inversely associated with preterm birth at<32 weeks and<34 weeks of gestation. Conclusions CL measured by transvaginal ultrasound in the second and the third trimester is a good predictor for preterm birth of twin pregnancy. CL≤36.5 mm and≤33.5 mm at 20-24 weeks of gestation could predict preterm birth<32 weeks and<34 weeks respectively. CL≤17.5 mm and≤18.5 mm at 28-32 weeks of gestation could predict preterm birth<32 weeks and<34 weeks respectively.