Oxidized Carbon dots, prepared from the acid dehydration of cellulose, were used in the preparation of self-assembled films with the biopolymer chitosan. The morphology, thickness and molecular interactions of the films were characterized with a variety of techniques including AFM, FTIR, UV-Vis and Surface Plasmon Resonance (SPR) spectroscopy. We noticed an increase in the thickness of the bilayers with increasing ionic strength and pH provided by the increase of the amount of CD and chitosan deposited in each cycle. This increase was mainly due to higher flexibility of the biopolymer on these conditions and to the formation of agglommerates of carbon dots in high ionic strength. On the other hand, the greater flexibility together with the higher adsorption have generated films with smoother morphology due to the higher coating on the spherical particles. The results described here suggest that the structure of the film, i.e., morphology and thickness can be easily controlled and reproduced, by controlling the conditions of the biopolymer and CD solutions, in addition to the traditional number of dips. Finally, the prepared films have potential application in the development of imunosensors, functional coatings, surface photoactive and UV protection films. (C) 2016 Elsevier B.V. All rights reserved.