The interaction of chitosan with bio-membranes, which plays important role in deciding its use in biological applications, is realized by investigating the interaction of chitosan with stearic acid (fatty acid) in Langmuir monolayers (at air water interface) and Langmuir-Blodgett (LB) films (after transferring it onto solid substrate). It is found from the pressure-area isotherms that the chitosan insertion causes an expansion of chitosan-fatty acid hybrid monolayers, which reduces the elasticity and make the film heterogeneous. It is likely that at low surface pressure chitosan is situated at the interface, interacting with stearic acid molecules via electrostatic and hydrophobic interactions whereas at high pressure chitosan mainly located at subsurface beneath stearic acid molecules. In the latter case the interaction is predominantly electrostatic yielding very small contribution to the surface pressure. The reduction of temperature of the subphase water allows more number of chitosan molecules to reach surface to increase the pressure/interaction. On the other hand, although pure chitosan is found difficult to relocate on the substrate from air-water interface due to its hydrophilic-like nature, it alongside stearic acid (amphiphilic molecules) can be transferred onto substrate using LB technique as evident from infrared spectra. Their out-of plane and in-plane structures, as extracted from two complementary surface sensitive techniques- X-ray reflectivity and atomic force microscopy, are found strongly dependent on the chitosan mole fraction and the deposition pressure. These analysis of the film-structure will essentially allow one to model the system better and provide better insight into the interaction. (C) 2017 Elsevier Inc. All rights reserved.