The fundamental aspect of cell-free synthesis systems is the in vitro transcription-translation process. By artificially providing the components required for protein expression, in vitro protein production alleviates various limitations tied to in vivo production, such as oxygen supply and nutrient constraints, thus showcasing substantial potential in engineering applications. This article presents a comprehensive review of cell-free synthesis systems, with a primary focus on biosensing and biomanufacturing. In terms of biosensing, it summarizes the recognition-response mechanisms and key advantages of cell-free biosensors. Moreover, it examines the strategies for the cell-free production of intricate proteins, including membrane proteins and glycoproteins. Additionally, the integration of cell-free metabolic engineering approaches with cell-free synthesis systems in biomanufacturing is thoroughly discussed, with the expectation that biotechnology will embrace greater prosperity.