The food-energy-water (FEW) nexus is interconnected and interdependent and provides a physical foundation for mankind. The production of safe food, renewable energy, and clean water through biological means, especially microbial bioconversion, has attracted an enormous attention worldwide. Recently, in vitro synthetic enzymatic biosystems (ivSEBs) comprised of numerous enzymes and coenzymes, as a disruptive biomanufacturing platform, has been proposed and demonstrated to address key challenges at the interface of the FEW nexus. Light, electricity, and hydrogen can provide energy to fix CO2 and produce food and biomass. Lignocellulose-derived cellulose can be converted to starch and biofuels. Starch can be further converted to bioenergy, including electricity, hydrogen and liquid fuels. These high-energy efficient bioprocesses lead to significantly less water usage and also can be used to reduce water pollution. In this review, the conceptual framework and latest advances of ivSEBs in the FEW nexus are summarized. Their limitations and future research directions on the design and improvement of ivSEBs are also discussed.