The trauma of central nervous system (CNS) can lead to glial scar, and it can limit the regeneration of neurons at the injured area, which is considered to be a major factor affecting the functional recovery of patients with CNS injury. At present, the study of the glial scar model in vitro is still limited to two-dimensional culture, and the state of the scar in vivo cannot be well mimicked. Therefore, we use a collagen gel and astrocytes to construct a three-dimensional (3D) model in vitro to mimic natural glial scar tissue. The effects of concentration changes of astrocytes on cell morphology, proliferation, and tissue performance were investigated. After 8 days of culture in vitro, the results showed that the tissue model contracted, with a measured shrinkage rate of 4.5%, and the compressive elastic modulus increased to nearly 4 times. Moreover, the astrocytes of the 3D tissue model have the ability of proliferation, hyperplasia, and formation of scar clusters. It indicates that the model we constructed has the characteristics of glial scar tissue to some extent and can provide an in vitro model for the research of glial scar and brain diseases.