It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on aminosilanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degreesC and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.