In total, 115 pregnant women underwent detection of Neutrophil Elastase (NE) and Fetal Fibronectin (fFN), and measurement of Cervical Length (CL) 20-34 weeks into pregnancy. The clinical value of these values in predicting preterm birth was analysed retrospectively. In the 29 women who gave birth before 34 weeks, the CL value (21.3 ± 9.8 mm) and the fFN and NE rates (65.5% and 75.9%, respectively) were significantly different compared with women undergoing childbirth after 34 weeks (P<0.05). The combined detection rate of CL and fFN was 78.6%, CL and NE was 84.4%, and fFN and NE was 88.9%. Moreover, the combined detection rate of CL, NE and fFN was 100%. The predictive value of premature labor in twin pregnancy using multiple markers was more successful than that of a single marker; therefore, these multiple markers could be used to evaluate clinical intervention.