Cancer stem cells (CSCs) are proposed to be responsible for tumor recurrence, metastasis and the high mortality rate of cancer patients. Isolation and identification of CSCs is crucial for basic and preclinical studies. However, as there are currently no universal markers for the isolation and identification of CSCs in any type of cancer, the method for isolating CSCs from primary cancer tissues or cell lines is costly and ineffective. In order to establish a reliable model of cervical cancer stem cells for basic and preclinical studies, the present study was designed to enrich cervical cancer CSCs using a nonadhesive culture system and to characterize their partial stemness phenotypes. Human cervical cancer cells (HeLa) were cultured using a nonadhesive culture system to generate tumor spheres. Their stemness characteristics were investigated through colony formation, tumor sphere formation, self-renewal, toluidine blue staining, chemoresistance, invasion assays, reverse transcription-polymerase chain reaction, immunofluorescence staining of putative stem cell markers, including octamer-binding transcription factor 4, SRY-box 2 and aldehyde dehydrogenase 1 family, member A1, and adipogenic differentiation induction. Typical tumor spheres were formed within 5-7 days under this nonadhesive culture system. Compared with the adherent parental HeLa cells, the colony formation capacity, self-renewal potential, light cell population, cell invasion, chemoresistance and expression of putative stem cell markers of the tumor sphere cells increased significantly, and a subpopulation of tumor sphere cells were induced into adipogenic differentiation. Using the nonadhesive culture system, a reliable model of cervical cancer stem cells was established, which is inexpensive, effective and simple compared with the ultra-low attachment serum free culture method. The stemness characteristics of the tumor sphere HeLa cells mirrored the CSC phenotypes. This CSC model may be useful for basic and preclinical studies of cervical cancer and other types of cancer.