In this work, layer by layer (LbL) thin film nanocomposite (TFN) membranes were prepared and used as pervaporation membranes to separate methyl tertiary butyl ether (MTBE)/methanol (MeOH) mixture. TFN membranes, comprising of carbon nanotubes (CNT), were prepared by interfacial polymerization (IP) of m-phenylenediamine (MPD) and trimesoyl chloride (TMC) monomers on the porous polyethersulfone/polyethylene glycol (PES/PEG) sublayer. Carbon nanotubes were functionalized by ethylenediamine (EDA) and incorporated into the polyamide (PA) layer at 0.01–0.04 wt.% loadings. After that, the surface roughness, surface hydrophilicity, and morphology of the resulting membranes were characterized. Also, the effect of the sequence of reactants deposition and concentrations of the nanotubes on the membrane performance were investigated using pervaporation of MTBE/MeOH under feed temperature and feed flow rate of 30 °C and 4 L.h-1, respectively. The pressure of permeate-side was about 100 mbar. Obtained results showed that both separation factor and permeation flux of LbL TFN membranes increased by adding CNT into the PA layer up to 0.02 wt.%. By increasing CNT loading to 0.04 wt.%, the separation factor decreased from 106 to 77.6, while permeation flux increased from 0.56 to 0.81 kg.m-2.h-1. It should be noted that, in the single-layer membrane, the separation factor decreased from 73.2 for TFC membrane to 56.4 for TFN membrane with increasing the CNT loading from the beginning. According to these results, LbL TFC and TFN membranes are expected to be the next generation of high-performance PV membranes.