Electrospinning is a common and effective technology used for the fabrication of biomimetic nanofibers targeting tissue regeneration applications. As for cartilage regeneration, nanofibers containing natural components derived from cartilage extracellular matrix (ECM) are preferred. However, it is not easy an task to electrospin glycosaminoglycan (GAG) like hyaluronic acid (HA) and chondroitin sulfate (CS) by themselves. In this study, HA and/or CS were co-electrospun with poly(L-lactide) (PLLA) or PLLA/gelatin (1:1 in weight ratio) to obtain GAG-containing composite nanofibers. All the prepared composite nanofibers were non-cytotoxic, able to support cell attachment, spread and proliferation. In the differentiation studies, the PLLA/GAG and the PLLA/gelatin/GAG nanofibers demonstrated stronger capacities in promoting the chondrogenic differentiation of both the bone marrow mesenchymal stromal cells (BMSCs) and chondrocytes than the respective PLLA and PLLA/gelatin nanofibers, even in the proliferation medium without extra inductive factors. The incorporation of gelatin greatly improved the hydrophilicity of the fibrous meshes. At the meantime, the PLLA/gelatin/GAG nanofibers were more efficient than the PLLA/GAG nanofibers in enhancing the chondrogenic differentiation. It was found that the PLLA/gelatin/HA/CS (HA and CS in 1:1 weight ratio) nanofibers demonstrated a stronger synergetic effect on up-regulating chondrogenesis than both the PLLA/gelatin/HA and the PLLA/gelatin/CS nanofibers, when the GAG amounts in all the preparations were controlled as 3 wt.%. Herein, GAG-containing composite nanofibers were successfully electrospun and their potentials for cartilage repair were proved.