In this study, a chitosan substrate was modified by simulated body fluid (SBF) treatment, in which the effect of the chosen crosslinking agent was investigated. Two crosslinking agents, glutaraldehyde (GA) and sodium tripolyphosphate (TPP), were used before the SBF process. By using TPP as the crosslinking agent, the Ca/P ratio and the degree of crystallinity were very close to the natural bone matrix. On the contrary, the substrate properties were very different from natural bone when the crosslinking agent GA was used. The results indicate that the produced substrates were biomimetic when the TPP was applied. On the SBF-modified chitosan substrates with TPP crosslinking, the cultured osteoblastic cells expressed better proliferation, mitochondria activity and differentiation ability. The chitosan crosslinked using TPP was a good template in the SBF process, which resulted in a highly biomimetic layer. This biomimetic substrate possesses excellent biocompatibility and osteoconduction ability, promising high potential in the promotion of bone tissue engineering.