Abstract: In this study, an alginate/chitosan bilayer composite film loaded with zinc oxide nanoparticles was fabricated by layer-by-layer casting method. Then, the effects of zinc oxide nanoparticles on the properties of bilayer composite film were studied by measuring mechanical, barrier, and optical characteristics. Moreover, the structural properties of film were characterized by atomic force microscope, scanning electron microscope, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The results showed that after incorporation of zinc oxide nanoparticles into the alginate/chitosan bilayer film, its tensile strength increased. Moreover, the elongation at break, water solubility, water vapor permeability, and transmittance decreased, whereas the color of appearance did not change significantly. Referring to the microstructure of bilayer film, with the concentration of nano zinc oxide ranging from 0.25% (w/w) to 1.00% (w/w), agglomeration of particles appeared on the surface of the alginate/chitosan bilayer composite film, and the roughness Ra increased from 3.12 nm to 3.53 nm. Infrared and thermogravimetric analysis indicated that there was a strong hydrogen-bonding interaction of nano zinc oxide with chitosan and alginate, improving the thermal stability of film. Therefore, the alginate/chitosan bilayer composite film loaded with nano zinc oxide has better packaging properties and applicability.