Chitosan, a biocompatible material that has been widely used in bone tissue engineering, is believed to have a high affinity to osteoblastic cells. This research is the first to prove this hypothesis. By using atomic force microscopy (AFM) with a chitosan-modified cantilever, quantitative evaluation of the interforce between chitosan and cells was carried out. A chitosan tip functionalized with Arg-Gly-Asp (RGD) was also used to measure the interforce between RGD-chitosan and osteoblastic cells. This research concluded by examining cell adhesion and spreading of chitosan substrates as further characterization of the interactions between cells and chitosan. The force measured by AFM showed that the interforce between chitosan and osteoblasts was the highest (209 nN). The smallest adhesion force (61.8 nN) appeared between chitosan and muscle fibroblasts, which did not demonstrate any osteoblastic properties. This result proved that there was a significant interaction between chitosan and bone cells, and correlated with the observations of cell attachment and spreading. The technique developed in this research directly quantified the adhesion between chitosan and cells. This is the first study to demonstrate that specific interaction exists between chitosan and osteoblasts.