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This work presents a layer?by?layer assembly strategy to generate polysaccharide multilayers on the surface of an all?liquid microfluidic chip, significantly enhancing the mechanical properties of the microchannel, and offering a biocompatible microenvironment for enzyme immobilization. All?liquid microfluidic enzymatic or cascade reactors are constructed, and the crucial role of polysaccharide multilayers on enhancing the enzyme loading and catalytic efficiency is demonstrated.Enzyme immobilization in the confines of microfluidic chips, that promote enzyme activity and stability, has become a powerful strategy to enhance biocatalysis and biomass conversion. Here, based on a newly developed all?liquid microfluidic chip, fabricated by the interfacial assembly of nanoparticle surfactants (NPSs) in a biphasic system, a layer?by?layer assembly strategy to generate polysaccharide multilayers on the surface of a microchannel, greatly enhancing the mechanical properties of the microchannel and offering a biocompatible microenvironment for enzyme immobilization, is presented. Using horseradish peroxidase and glucose oxidase as model enzymes, all?liquid microfluidic enzymatic and cascade reactors have been constructed and the crucial role of polysaccharide multilayers on enhancing the enzyme loading and catalytic efficiency is demonstrated.