Occurring preterm birth (PTB) before 37 weeks of pregnancy is one of major causes, resulting in perinatal mortality and neonatal morbidity. Nevertheless, the proteomic study on the development of biomarker(s) for early detection of PTB, unfortunately, has not been matured. In this study, we performed shotgun proteomic analysis of the cervicovaginal fluid (CVF) samples from PTB and its normal counterpart, so as to unveil the protein biomarkers using two dimension-nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-nLC-ESI-MS/MS). The quantitative assessments of CVF proteome from both PTB and control were carried out through an isobaric labeling approach with iTRAQ. Out of 1044 proteins identified, 1009 proteins were able to be in common quantified in PTB/control CVF samples. From MS/MS-based isobarically quantitative determination, 30 proteins from PTB were twofold or more upregulated, compared to that of control ones, while 59 proteins were measured to be twofold decreased. Based on the bioinformatic analyses (e.g., Gene Ontology and KEGG), we found that 18 proteins, having the different expression levels between PTB and control, were implicated in immune response in cell. Resultingly, both of up-/down-regulated proteins identified in this study might be applicable to the development of biomarker(s) having the potential in the clinical diagnosis of PTB.