We report a biomimetic polyelectrolyte based on amphiphilic polymer nanosheet multilayer films. Copolymers of poly(N-dodecylacrylamide-co-vinylphosphonic acid) [p(DDA/VPA)] form a uniform monolayer at the air-water interface. By depositing such monolayers onto solid substrates using the Langmuir-Blodgett (LB) method, multilayer lamellae films with a structure similar to a bilayer membrane were fabricated. The proton conductivity at the hydrophilic interlayer of the lamellar multilayer films was studied by impedance spectroscopy under temperature- and humidity-controlled conditions. At 60 °C and 98% relative humidity (RH), the conductivity increased with increasing mole fraction of VPA (n) up to 3.2 × 10-2 S cm-1 for n = 0.41. For a film with n = 0.45, the conductivity decreased to 2.2 × 10-2 S cm-1 despite the increase of proton sources. The reason for this decrease was evaluated by studying the effect of the distance between the VPAs (lVPA) on the proton conductivity as well as their activation energy. We propose that for n = 0.41, lVPA is the optimal distance not only to form an efficient two-dimensional (2D) hydrogen bonding network but also to reorient water and VPA. For n = 0.45, on the other hand, the lVPA was too close for a reorientation. Therefore, we concluded that there should be an optimal distance to obtain high proton conductivity at the hydrophilic interlayer of such multilayer films.