Objective: To investigate the factors affecting spontaneous preterm birth in singleton pregnancy, so as to provide insights into reducing the risk of preterm birth. Methods: A total of 230 lying-in women with spontaneous preterm birth in singleton pregnancies admitted to the Hangzhou Obstetrics and Gynecology Hospital were selected as the case group, and lying-in women with full-term birth in singleton pregnancies in the hospital during the study period were selected as controls. Basic information and pregnancy status were collected through the hospital information system, and factors affecting spontaneous preterm birth in singleton pregnancies were analyzed using a multivariable logistic regression model. Results: The lying-in women in the case group had a mean age of (33.40±3.16) years and a mean gestational weeks at delivery of (34.72±2.15) weeks. The lying-in women in the control group had a mean age of (28.30±3.92) years and a mean gestational weeks at delivery of (39.84±2.09) weeks. Multivariable logistic regression analysis identified age of 35 years and older (OR=1.280, 95%CI: 1.022-1.603), induced abortion three times and above (OR=3.122, 95%CI: 1.368-7.121), history of preterm birth (OR=3.769, 95%CI: 1.725-8.240), premature rupture of membranes (OR=12.708, 95%CI: 4.836-33.391), gestational hypertension (OR=2.934, 95%CI: 1.313-6.550), gestational diabetes (OR=2.510, 95%CI: 1.249-5.038) and cervical canal length ≤25 mm (OR=5.832, 95%CI: 2.380-14.279) as factors affecting spontaneous preterm birth in singleton pregnancies. Conclusion Spontaneous preterm birth in singleton pregnancy may be associated with age, number of induced abortions, premature rupture of membranes, complications in pregnancy and cervical canal length.