Objective: To construct an in vitro lung cancer model using 3D bioprinting and evaluate the feasibility of the model. Transcriptome sequencing was used to compare the differential genes and functions of 2D and 3D lung cancer cells. Methods: 1. A549 cells were mixed with sodium alginate/gelatine/fibrinogen as 3D-printed biological ink to construct a hydrogel scaffold for the in vitro model of lung cancer; 2. A hydrogel scaffold was printed using a extrusion 3D bioprinter; 3. The printed lung cancer model was evaluated in vitro; and 4. A549 cells cultured in 2D and 3D tumour models in vitro were collected, and RNA-seq conducted bioinformatics analysis. Results: 1. The in vitro lung cancer model printed using 3D-bioprinting technology was a porous microstructure model, suitable for the survival of A549 cells. Compared with the 2D cell-line model, the 3D model is closer to the fundamental human growth environment; 2. There was no significant difference in cell survival rate between the 2D and 3D groups; 3. In the cell proliferation rate measurement, it was found that the cells in the 2D group had a speedy growth rate in the first five days, but after five days, the growth rate slowed down. Cell proliferation showed a declining process after the ninth day of cell culture. However, cells in the 3D group showed a slow growth process at the beginning, and the growth rate reached a peak on the 12th day. Then, the growth rate showed a downward trend; and 4. RNA-seq compared A549 cells from 2D and 3D lung cancer models. A total of 3112 genes were differentially expressed, including 1189 up-regulated and 1923 down-regulated genes, withp-value ≤ 0.05 and |Log2Ratio| ≥ 1 as screening conditions. After functional enrichment analysis of differential genes, these differential genes affect the biological regulation of A549 cells, thus promoting lung cancer progression. Conclusion: This study uses 3D-bioprinting technology to construct a tumour model of lung cancer that can grow sustainably in vitro. Three-dimensional bioprinting may provide a new research platform for studying the lung cancer TME mechanism and anticancer drug screening.