AbstractThe development of cascade cell‐free systems reduces the requirement for extensive metabolic engineering and optimization to increase in vivo pathway flux. For continuous operation and increased stability, direct enzyme entrapment during reactor fabrication by three‐dimensional (3D)‐printing allows for simple immobilization procedures without enzyme‐specific optimization. In this study, the isopentenol utilization pathway (IUP) was selected for the synthesis of amorphadiene, an antimalaria drug precursor, using a 3D‐printed, sequentially immobilized, microfluidic reactor. As an initial proof‐of‐concept, alkaline phosphatase (ALP) was entrapped in a poly(methyl methacrylate) (PMMA)‐based matrix during stereolithographic 3D‐printing and was kinetically characterized. No significant shift of the kinetically modeled substrate binding affinity was observed during immobilization and continuous operation of an entrapped ALP microfluidic reactor displayed high stability. The IUP enzymes retained moderate activity during entrapment (6.6%–9.6%) relative to the free enzyme solutions, however the sequentially immobilized IUP microfluidic reactor was severely limited by low pathway flux due to the use of stereolithographic 3D‐printing which significantly diluted enzyme concentrations for printing. Although this study demonstrated the use of additive manufacturing for the synthesis of amorphadiene using a complex five‐enzyme cascade microfluidic reactor, stereolithographic enzyme entrapment remains limited in scope and dependent on advancements to additive manufacturing technologies.