The poor intrinsic mechanical properties of chitosan hydrogels have greatly hindered their practical applications. Inspired by nature, we proposed a strategy to enhance the mechanical properties of chitosan hydrogels by construction of a nanofibrous and cellular architecture in the hydrogel without toxic chemical crosslinking. To this end, electrospun nanofibers including cellulose acetate, polyacrylonitrile, and SiO nanofibers were introduced into chitosan hydrogels by homogenous dispersion and lyophilization. With the addition of 30% cellulose acetate nanofibers, the cellular structure could be maintained even in water without crosslinking, and integration of 60% of the nanofibers could guarantee the free-standing structure of the chitosan hydrogel with a low solid content of 1%. Moreover, the SiO nanofiber-reinforced chitosan (SiO NF/CS) three-dimensional (3D) matrices exhibit complete shape recovery from 80% compressive strain and excellent injectability. The cellular architecture and nanofibrous structure in the SiO NF/CS matrices are beneficial for human mesenchymal stem cell adhesion and stretching. Furthermore, the SiO NF/CS matrices can also act as powerful vehicles for drug delivery. As an example, bone morphogenetic protein 2 could be immobilized on SiO NF/CS matrices to induce osteogenic differentiation. Together, the electrospun nanofiber-reinforced 3D chitosan matrices exhibited improved mechanical properties and enhanced biofunctionality, showing great potential in tissue engineering.