The multienzyme complex system has become a research focus in synthetic biology due to its highly efficient overall catalytic ability and has been applied to various fields. Multienzyme complexes are formed by cascading complexes, which are multiple functionally related enzymes that continuously and efficiently catalyze the production of substrates. Compared with current mainstream microbial cell catalytic systems, in vitro multienzyme molecular machines have many advantages, such as fewer side reactions, a high product yield, a fast reaction speed, easy product separation, a tolerable toxic environment, and robust system operability, showing increasing competitiveness in the field of biomanufacturing. In this review, the research progress of multienzyme complexes in nature and multienzyme cascades in vivo or in vitro will be introduced, and the discovered enzyme cascades concerning scaffolding proteins will also be discussed. This review is expected to provide a more theoretical basis for the modification of multienzyme complexes and broaden their application in the field of synthetic biology.