Novel flexible NO2 gas sensors which, can function at room temperature, were formed by the layer-by-layer (LBL) self-assembly of multi-walled nanotubes (MWCNTs) on plastic substrates. First, a negatively charged substrate was prepared by the formation of an organic monolayer (3-mercapto-1-propanesulfonic acid sodium salt; MPS) on a polyester (PET) substrate with a pair of comb-like Au electrodes. Then, two-cycle poly(4-styrenesulfonic acid-co-maleic acid)/poly(allylamine hydrochloride) (PSSMA/PAH) bilayers were deposited on an MPS-modified substrate. Finally, MWCNT multilayers were formed as alternating layers of cationic PAH and negatively charged MWCNTs on the modified substrate. Changes in charge density and the conformation of PSSMA at various pH conditions, caused the bilayer (PSSMA/PAH) that was prepared at pH 4 to be more flexible than that prepared at pH 8. The (MWCNTs/PAH)10 multilayer thin film sensor was very flexible (there was only a 2% deviation in sensitivity when the sensor was bent at a downward angle of up to 60°), and high sensitivity, good linearity (R2 = 0.9716), a rapid response and an undesirable cross-sensitivity effect. The detachment of adsorbed gas molecules by irradiation under UV was studied at room temperature.