The production of biofuels from renewable sugars isolated from plants or produced through artificial photosynthesis would provide a sustainable transportation fuel alternative for decreasing reliance on crude oil, mitigating greenhouse gas emissions, creating new manufacturing jobs, and enhancing national energy security. Since sugar costs usually account for at least 50% of biofuels’ selling prices, it is vital to produce desired biofuels with high product yields and at low production costs. Here I suggest high-product yield and potentially low-cost biofuels production through cell-free synthetic enzymatic pathway biotransformation (SyPaB) by in vitro assembly of stable enzymes and (biomimetic) coenzymes. SyPaB can achieve high product yields or high energy efficiencies that living entities cannot achieve. Great potentials of SyPaB, from chiral compounds, biodegradable sugar batteries, sulfur-free jet fuel, hydrogen, sugar hydrogen fuel cell vehicles, high-density electricity storage, to synthetic starch, are motivation to solve the remaining obstacles by using available technologies, such as protein engineering, enzyme immobilization, unit operations, and technology integration. The biotransformation through in vitro assembly of numerous enhanced-performance and stable enzymes in one bioreactor that can last a very long reaction time (e.g., several months or even years) would be an out-of-the-box solution for high-yield and low-cost biofuels production.