Abstract A novel triple-layer proton exchange membrane comprising two thin layers of structurally modified chitosan, as methanol barrier layers, both sides coated with Nafion®105 is prepared and tested for high-performance direct methanol fuel cell applications. A tight adherence is detected between layers from SEM and EDX data for the cross-sectional area of the newly designed membrane, which are attributed to high affinity of opposite charged polyelectrolyte layers. Proton conductivity and methanol permeability measurements show improved transport properties for the multi-layer membrane compared to Nafion®117 with approximately the same thickness. Moreover, direct methanol fuel cell tests reveal higher open circuit voltage, power density output, and overall fuel cell efficiency for the triple-layer membrane than Nafion®117, especially at concentrated methanol solutions. A power output of 68.10 mW cm−2 at 5 M methanol feed is supplied using multi-layer membrane, which is found to be about 72% more than that of for Nafion®117. In addition, fuel cell efficiency for multi-layer membrane is measured about 19.55% and 18.45% at 1 and 5 M methanol concentrations, respectively. Owing to the ability to provide high power output, significantly reduced methanol crossover, ease of preparation and low cost, the triple-layer membrane under study could be considered as a promising polyelectrolyte for high-performance direct methanol fuel cell applications.