Abstract: In this work, a trilayer composite film of alginate-polyvinylpyrrolidone-alginate loaded with β-lactoglobulin nanoparticles was prepared based on the layer-by-layer assembly. The formation of β-lactoglobulin nanoparticles was induced by adjusting the ambient pH and temperature in combination with binding apigenin. The formation pattern and storage stability of β-lactoglobulin nanoparticles were investigated by particle size, polydispersity and Zeta potential. Then, the β-lactoglobulin nanoparticles were loaded into sodium alginate-polyvinylpyrrolidone-sodium alginate trilayer composite film to investigate the effects of nano-loading on the mechanical, transmission, optical and thermal properties of the film. The results showed that β-lactoglobulin nanoparticles with good stability could be obtained by adjusting the ambient pH to 7.1 and heating the temperature to 75 ℃ under the protein/ligand molar ratio of 1:8. The mechanical properties and water vapor barrier of the films were significantly improved when the nanoparticles were added to the trilayer composite films at concentrations of 0.2 and 0.3 mg/mL. Moreover, the addition of β-lactoglobulin nanoparticles improved the light transmittance and thermal stability of the trilayer composite film. In conclusion, the trilayer composite film made of alginate loaded with β-lactoglobulin nanoparticles exhibited good packaging properties and high application potential.