Chitosan (C) and zein (Z) were used to develop bilayer films with a characteristic one-way water barrier using a layer-by-layer (LBL) casting method. The effects of mass ratios (C:Z1:1, C:Z1:2, C:Z1:3, C:Z3:1, C:Z2:1) on the microstructure and physicochemical properties of bilayer films were investigated. Bilayer films had uniform microstructures, and C:Z = 1:3 showed a firmer structure as the Z aggregates were distributed in the continuous phase of C. The intermolecular interactions between the C and Z layers were observed using FTIR and XRD analysis. TGA demonstrated that adding Z layer enhanced the thermal stability of C films. LBL coating gave the C/Z bilayer film an increased elongation and tensile strength, as well as a decreased water vapor and oxygen permeability, especially for C:Z = 1:3 which had better properties. The results suggested that C and Z bilayer films may be a promising material for food packaging with the desired water resistance.