In order to promote the growth of chondrocyte ATDC-5 in collagen type II-hyaluronic acid-chondroitin sulfate composite scaffolds constructed previously in vitro, the sustained-releasing chitosan microspheres loading TGF-β1 were prepared by emulsification and cross-linking. In addition, ATDC-5 was inoculated into the scaffolds incorporating the chitosan microspheres with TGF-β1. Results show that the morphology of microsphere was round and uniform, mean diameter was about 100 nm, absorption rate was up to 983.7%±4.38%.When the microsphere was incubated under the condition of 107 U/L lysozyme, the degradation rate was only 51.0%±1.8% on day 28. Moreover, to compare the effect of TGF-β1, the growth of ATDC-5 in different scaffolds was observed by MTT assay and fluorescence staining test. According to the cumulative release curve, TGF-β1 was released quickly at initial 24 h, then gradually decelerated, finally reached the plateau after 120 h. MTT assay and fluorescence staining test demonstrated that the scaffolds were suitable for ATDC-5 growth and proliferation, as well as, suggested that the sustained-releasing chitosan microspheres loading TGF-β1 could significantly promote the growth of ATDC-5.