The architecture and biomaterial are vital for three-dimensional culture of cells in scaffolds, so collagen-chitosan scaffolds suitable for the proliferation of adipose tissue-derived stem cells (ADSCs) were fabricated in this study. Chitosan was fully mixed with collagen with different volume ratio and cross-linked. The microstructure, pore size, bibulous ability, water content, interval porosity, enzyme degradation and affinity were examined before and after cross-linking. During ADSCs cultured in scaffold, the viability and metabolic rates were measured. After 14 days, the surface markers, specific transcription factors and multi-differentiation potential were assayed to identify the stemness of expanded cells. According to the pore size, bibulous ability, interval porosity, degradation rate and affinity of the scaffold, we chose cross-linked scaffolds of 7:3 material ratio as a better scaffold for ADSCs proliferation, and ADSCs could be expanded by more than 20 times. All expanded cells still maintained stem cell characteristics and pluripotency. So our developed collagen-chitosan scaffolds can promote ADSCs adhesion, expansion, and maintain pluripotency.