Despite the explosive interest in the biomass-based nanoparticles, cellulose nanocrystal (CNC) monolayers are rarely reported. Here, ultrathin CNC layers were prepared on the chemically modified substrate using Langmuir-Blodgett technique at air/water interface. Since the surface of sulfated CNC was negatively charged, they could bound to the substrates whose surface was modified positively. The substrates including glass and polymers were modified with positive surfactant and the selective binding of CNCs to the surface was verified by imaging tools. Surface pressure-area (π-A) isotherm curves of CNC were obtained at the air/water interface according to the CNC concentrations. The gaseous, extended liquid and condensed solid phases were observed with the π-A isotherm curves and the specific surface pressure was determined for the formation of solid phase CNC layers. The ultrathin CNC layer was confirmed with SEM, which showed well-organized crystalline domains in the compact layer.