Sulfated polysaccharides have received much attention in recent years due to their special biological activities, especially the regulation of the biological activity of growth factors such as the representative inductive growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2). However, the regulatory mechanisms from the aspect of the molecular chain structure have rarely been reported. In this article, we selected three kinds of sulfonates containing different backbone structures and functional groups, 2-N,6-O-sulfated chitosan (26 SCS), sulfated dextran (DSS) and poly(sodium-p-styrenesulfonate) (PSS), to explore the interaction between them and rhBMP-2. From in vivo and in vitro osteogenesis-related experiments, 26 SCS showed the best promoting effect on rhBMP-2 induced osteogenic differentiation and the sulfated amino group in 26 SCS could specifically bind to rhBMP-2. These findings indicated that the polysaccharide chain structure was a prerequisite for the synergy effect between 26 SCS and rhBMP-2; the effective combination of -SO3- and rhBMP-2 was an important factor in protecting the bioactivity of rhBMP-2. In addition, the presence of the sulfated amino group was the key factor in the specific binding between 26 SCS and rhBMP-2 and provided the possibility of capturing factors in vivo.