Abstract: Objective: To highlight the developmental process of 3D cell culture technology system, which is more suitable for isolating and identifying lung cancer stem-like cells than 2D cell culture technology system, and to explore the application of 3D cell cultures in the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer. Methods: Cells (104 /well) from the human lung adenocarcinoma cell lines A549 and RPMI 1640 were cultured in complete medium containing 10% fetal bovine serum. Cell suspension was cultured in a BME basal medium. A growth curve was drawn after 7 d of culture. The stem-like cell was identified through a mammosphere culture, drug resistance and invasion assay, and flow cytometry. Data of A549 cells cultured in 3D and 2D traditional cell culture technologies were compared. Results: Cells from the 3D cell culture had higher tumor formation rates (20.75± 0.85) d vs. (60.25±1.49) d, P < 0.01) and tumor sphere formation (28.50%±1.17% vs. 8.67%±0.80%, P < 0.01) than those from the 2D cell culture. Moreover, cells from 3D cell culture were more invasive and resistant to therapy (58.17%± 2.19% vs. 41.70%±5.81% in 48 h, P < 0.01; 33.27%±5.76% vs. 27.30%±4.25% in 72 h, P < 0.01). Phenotype experimental results demonstrated that the CD44 and CD326 cells were double-positive, whereas the CD24 cell was negative. Conclusion: The proportion of stem-like cells in A549 cell line after 3D cell culture significantly increased compared with 2D cell culture. The 3D cell culture can promote the proliferation of lung cancer stem cells.