: In vitro multienzymatic bioreaction systems are attracting increasing attention for the development of bioproduction systems. The de-coupling of the biocatalytic pathway from the cellular machinery aimed at growth and survival allows achievement of high product yields and thus reduces byproduct or waste generation. Additionally, the use of several enzymes allows the realization of much more complex synthetic schemes, thus expanding the chemical diversity of synthetic compounds with new chemical properties or bioactivities. This chapter provides a survey of in vitro multienzymatic bioreaction systems used for biosynthesis, discusses process design aspects for the technical realization of multienzymatic bioreactions, highlights advantages and recent developments in using multienzyme microreactors, and finally reviews examples and strategies of the co-immobilization of multienzymes in nano/microsized materials.