Glaucoma is the world's first irreversible blinding eye disease, characterized by degenerative changes in retinal ganglion cells (RGCs), which have a significant impact on the global economy and health. The molecular and biological mechanisms of its pathological changes are still unclear. At present, glaucoma surgery and drug therapy are still limited to controlling the intraocular pressure in the normal range. Three-dimensional culture technology can enable cells to form a certain spatial structure in vitro, which is conducive to cell-cell and cell-environment interactions. Compared with traditional two-dimensional culture, three-dimensional culture technology is closer to the physiological environment of cells in vivo, which is of great significance for the study of pathophysiological changes of diseases and high-throughput drug screening. This review discusses the application of trabecular mesh and three-dimensional culture of retinal cells in the basic research of glaucoma, aiming to provide new ideas for further exploring the pathophysiological mechanism of glaucoma.