The rapid development of synthetic biology has enabled the production of compounds with revolutionary improvements in biotechnology. DNA manipulation tools have expedited the engineering of cellular systems for this purpose. Nonetheless, the inherent constraints of cellular systems persist, imposing an upper limit on mass and energy conversion efficiencies. Cell-free protein synthesis (CFPS) has demonstrated its potential to overcome these inherent constraints and has been instrumental in the further advancement of synthetic biology. Via the removal of the cell membranes and redundant parts of cells, CFPS has provided flexibility in directly dissecting and manipulating the Central Dogma with rapid feedback. This mini-review summarizes recent achievements of the CFPS technique and its application to a wide range of synthetic biology projects, such as minimal cell assembly, metabolic engineering, and recombinant protein production for therapeutics, as well as biosensor development for in vitro diagnostics. In addition, current challenges and future perspectives in developing a generalized cell-free synthetic biology are outlined.