In recent years, the application of cell-free protein synthesis systems in biosensing has been developing rapidly. Cell-free synthetic biology, with its advantages of high biosafety, fast material transport, and high sensitivity, has overcome many defects of cell-based biosensors and provided an abiotic substitute for biosensors. In addition, the application of freeze-drying technology has improved the stability of such systems, making it possible to realize point-of-care application of field detection and broadening the application prospects of cell-free biosensors. However, despite these advancements, challenges such as the risk of sample interference due to the lack of physical barriers, maintenance of activity during storage, and poor robustness still need to be addressed before the full potential of cell-free biosensors can be realized on a larger scale. In this review, current strategies and research results for improving the performance of cell-free biosensors are summarized, including a comprehensive discussion of the existing challenges, future trends, and potential investments needed for improvement.