Biomimetic compartmentalization immobilization of multi-enzyme system is a frontier for in vitro synthetic biology, focusing on the spatial and temporal separation of reactions. Compared with simple co-immobilization, biomimetic compartmentalization immobilization can form substrate channels and promote the transmission of intermediates for sequential or coupling reaction. By controlling the relative positions of the enzymes on carriers, this method improves system stability, productivity, as well as purity of product. In this review, we summarized the recent advances of carriers for biomimetic compartmentalization immobilization of multi-enzyme systems, including metal-organic frameworks (MOFs), polymer vesicles and polymer capsules. Metal-organic frameworks (MOFs) are porous coordination materials which are composed of metal ions as nodes and organic linkers. MOFs possess unique characteristics including high porosity, large specific surface area and tunable structure, which are suitable for multi-enzyme systems. The strategies involving the hierarchically porous MOFs, MOF-on-MOF and multi-MOF combinations construct compartmentalized environments for efficient catalytic reactions in vitro. Polymer vesicles are hollow nanostructures composed of amphiphilic block copolymers. The membrane structure of polymer vesicles, similar to the natural phospholipid bilayers, has good mechanical stability and biocompatibility for protecting enzyme molecules, and provides unique microenvironment for sequential reactions. Multiple small vesicles were encapsulated into the larger vesicles to form a “vesicle-in-vesicle” by mimicking the structure of cellular organelles. Polymer capsules with a core-shell spherical nanostructure are formed by the templating method, and have structural stability and excellent shape controllability. Multilayered core-shell structures created by layer-by-layer self-assembly are applied for compartmentalized immobilization of multi-enzyme. In the future, the integration of microfluidic technologies with biomimetic compartmentalization immobilization of multi-enzyme is expected to provide highly efficient and stable multi-enzyme catalytic systems for in vitro synthetic biology and green biomanufacturing.