Objective To estimate the independent association of a short cervical length (CL), positive fetal fibronectin (fFN), amniotic fluid (AF) sludge, and cervical funneling with spontaneous preterm birth in twin pregnancies. Methods Retrospective cohort study of twin pregnancies managed by a single maternal-fetal medicine practice from June 2005 to February 2014. All patients underwent transvaginal sonographic CL and fFN testing. We reviewed all images from the first CL at 22(0/7) to 25(6/7) weeks for the presence of (1) a short CL, which is defined as ≤25 mm, (2) AF sludge, and (3) cervical funneling, and also recorded (4) the fFN result from that time. Image reviewers were blinded to pregnancy outcomes. Patients with cerclage were excluded. Using logistic regression, we calculated the independent association between these four biomarkers and spontaneous preterm birth. Results A total of 635 patients with twin pregnancies were included. The markers independently associated with spontaneous preterm birth <35 weeks were short CL (adjusted odds ratio [aOR]: 10.73; 95% confidence interval [CI]: 3.21-35.81), positive fFN (aOR: 3.25; 95% CI: 1.13-9.33), and AF sludge (aOR: 2.11; 95% CI: 1.04-4.27). Similarly, these three markers were independently associated with earlier gestational ages at delivery. Cervical funneling was not independently associated with spontaneous preterm birth <35 weeks nor gestational age at delivery. The risk of spontaneous preterm birth increased significantly with the number of positive biomarkers (short CL, positive fFN, and AF sludge). Conclusion In twin pregnancies, a short CL, positive fFN, and AF sludge are independently associated with spontaneous preterm birth. Cervical funneling is not independently associated with spontaneous preterm birth in twins.