AbstractPolyethylene terephthalate (PET) has been widely used in food packaging due to its excellent comprehensive properties, but has relatively low barrier properties towards small molecules. In this study, whey protein isolate (WPI)/chitosan (CS)/microcrystalline cellulose (MCC) solution was sprayed evenly on PET which pretreated by corona discharge (CD) and uniform glow discharge (UGD) plasma to prepare WPI/CS/MCC/PET bilayer film, and the properties, structure, and morphology of films were characterized. The water contact angle of UGD treated PET was significantly decreased by 79.01% due to increased polar functional groups. After coated with WPI/CS/MCC film, the reduced oxygen and carbon dioxide transmission rates of untreated and pretreated PET was in the range of 71.74–94.64%, and UGD pretreatment was more efficient to improve gas barrier properties than CD, but the mechanical and water vapor barrier properties of pretreated bilayer film weakened a little. There was a high intermolecular interaction between hydroxyl and amino groups of WPI/CS/MCC film and polar groups, such as COC and CH bonds, carbonyl, and methyl groups, of UGD‐pretreated PET, as verified by Fourier transform infrared with attenuated total reflection. In conclusion, UGD‐pretreated WPI/CS/MCC/PET bilayer film was significantly efficient to enhance the gas barrier properties of single‐layer PET.Practical ApplicationsPolyethylene terephthalate (PET) has relatively low barrier properties and its application has been limited in food high air tightness packaging. The present study aimed to develop biopolymer‐based high barrier bilayer film using surface‐pretreated PET substrate. The thermogravimetric analysis, mechanical, and barrier properties were investigated, and the structural characterization and morphology of bilayer films were investigated by Fourier transform infrared with attenuated total reflection analysis, environmental scanning electron microscopy, and atomic force microscope. This work provides a foundation for enhancing the gas barrier properties of single‐layer PET using the combination of biopolymer‐based coating and surface pretreatment.