The invention discloses a manufacturing method of a water and oxygen barrier layer. At least two thin film layers are successively manufactured on a substrate to form a water and oxygen composite barrier layer. When each thin film layer is manufactured, manufacturing parameters of at least two any adjacent thin film layers, which are provided and selected during manufacturing, are different so that each thin film layer in the water and oxygen composite barrier layer forms a disconnected mismatch structure of a water and oxygen microchannel. A plurality of thin film layers are manufactured on the substrate to form the water and oxygen composite barrier layer. Through adjusting one or more parameters influencing film deposition, the films can grow with different structures, different properties and even different growth modes so that a water vapor pathway in a previous film layer and a water vapor pathway in a next film layer are mismatched. The water vapor pathways which can not be connected can not conduct the water and oxygen in atmosphere so that water and oxygen barrier performance of the thin films is increased. The method is simple and easy to use. In future industrial production of a flexible display barrier layer, the method possesses considerable popularization potential.