Abstract: Multiwalled carbon nanotubes (MWCNTs) were treated in 1∶3 concentrated nitric-sulfuric acid to cut them into short tubes and to create carboxyl groups at their ends. Homogeneous multilayer films of the shortened MWCNTs were assembled on a silica glass substrate by a layer-by-layer method, based on electrostatic interaction of positively charged cationic polyelectrolyte chitosan (CS) and the negatively charged and shortened MWCNTs. The film assembly and characteristics of the CS/MWCNT multilayer films were investigated. The process of assembly was monitored through ultraviolet-visible spectroscopy, which indicated homogeneous and consecutive growth. Atomic force microscopy and scanning electron microscopy were used to characterize the surface of the films. The films showed stable optical properties and were promising as biosensors.