Astrocytes play a critical role in supporting the normal physiological function of neurons in the central nervous system (CNS). Astrocyte transplantation can potentially promote axonal regeneration and functional recovery after spinal cord injury (SCI). Fibrin and collagen hydrogels provide a growth-permissive substrate and serve as carriers for therapeutic cell transplantation into an injured spinal cord. However, the application of fibrin and collagen may be limited due to their relatively rapid degradation rate in vivo. In this study, immature astrocytes isolated from neonatal rats were cultured in fibrin hydrogel containing aprotinin and collagen hydrogel crosslinked with poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG), and the cell behavior in these hydrogels was studied. The cell viability of astrocytes in the hydrogels was tested using the LIVE/DEAD® assay and the AlamarBlue® assay, and this study showed that astrocytes maintained good viability in these hydrogels. The cell migration study showed that astrocytes migrated in the fibrin and collagen hydrogels, and the migration speed was similar in these hydrogels. Crosslinking of collagen hydrogel with 4S-StarPEG did not change the astrocyte migration speed. However, the addition of aprotinin to the fibrin hydrogel inhibited astrocyte migration. The expression of chondroitin sulfate proteoglycans (CSPG), including neural/glial antigen 2 (NG2), neurocan, and versican, by astrocytes grown in the hydrogels was analyzed by quantitative RT-PCR, and no significant difference was found.