<p>RNA scaffolds are synthetic noncoding RNA molecules designed with 3D folding structures to spatially organize protein in vivo. RNA scaffolds offer greater flexibility compared to DNA or protein scaffolding strategies, allowing for the adjustment of relative distances and spatial orientations between proteins. In this study, we utilized these characteristics of RNA scaffolds to achieve the efficient assembly of metabolic pathways, a key objective in synthetic biology and metabolic engineering. Using a cell-free protein synthesis (CFPS) system, we successfully optimized an RNA scaffold-based multi-enzyme pathway in an in vitro environment using a CFPS system. RNA scaffolds were employed to precisely organize the lycopene biosynthetic enzymes—geranylgeranyl pyrophosphate synthase, phytoene synthase, and phytoene desaturase—via fusion with specific RNA-binding domains. This strategic enzyme localization significantly enhanced the efficiency of substrate transfer and increased pathway productivity. To improve structural stability, human tRNA elements were incorporated into the RNA scaffold design, and RNase inhibitor was introduced to preserve the scaffold integrity over extended periods. The developed CFPS system enabled rapid testing of various RNA scaffold designs and enzyme combinations, facilitating the identification of optimal conditions for pathway assembly. This study demonstrates the potential of RNA scaffolds in CFPS systems for the optimization of multi-enzyme pathways, providing a versatile platform for studying intracellular metabolic pathways, advancing enzyme engineering, and enabling the efficient production of high-value bio-products.</p>