One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects.