The present study reports on the fabrication of new composite chitosan-based knitted three-dimensional textile scaffolds for regenerative medicine. A critical research objective remained the fabrication of pure chitosan microfiber yarn with defined size and directional alignment, which could lead to textile structure, for example, woven and/or knitted fabrics. The construction of such textile structure with pure chitosan fibers possessing adequate tensile strength was recently realized in our laboratory. In the combination of micro/nanofiber composites, one can benefit from both the strength provided by the woven/knitted structure of the microfiber and the cell-seeding ability of the spun nanofiber mesh. Therefore, chitosan knitted fabrics were functionalized with gelatin nanofibers producing new interfaces for cell seeding. Both, pure chitosan and chitosan/gelatin scaffolds were proved non-cytotoxic to fibroblasts. Moreover, in vitro test with osteoblasts culture was carried out in order to analyze the influence of the surfaces and interfaces of the functionalized textile scaffold on cell ingrowth and proliferation.