Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits. Cell-free gene expression systems have long been used to address fundamental research questions. Now, owing to technological advances, these systems are finding wider applications in the field of synthetic biology, including in biosensing, biomanufacturing, education and the design of gene networks.