With an aim to mimic natural extracellular matrix, we fabricated the nano- and microfibrous matrix with chitosan by electrospinning nanofibers onto predefined microfibrous mesh for effective chondrocytes cultivation. Rolling the double-layered nano-/microfibrous membranes produced three-dimensional (3-D) scaffolds that exhibited the interconnected open pore structure in their scanning electron microscopy images. In vitro chondrocyte culture experiment showed that this nano-/microfibrous 3-D matrix provided a significantly greater microenvironment for chondrocytes to proliferate and produce glycosaminoglycan as compared with only microfibrous 3-D matrix. This difference could be explained by the result on 2-D membrane, where chitosan nanofibrous surface substantially facilitated the cellular attachment and proliferation, and efficiently prevented phenotypic changes of chondrocytes, when compared with chitosan microfibrous membrane and film. In this regard, the nano-/microfibrous 3-D matrix we fabricated in this study would possess a great potential as a system for effective chondrocyte cultivation and also for application to cartilage regeneration therapy.