Cell-free protein synthesis (CFPS) systems based on crude cell extracts have been used in protein expression in vitro. With the researchers’ endeavor for decades, the CFPS system has been developed as an important research tool in many frontiers of fundamental and applied biology because of its clear genetic background and simplicity to control the reaction. The yield of CFPS systems derived from prokaryote or eukaryote has increased to several grams per liter with constantly decreasing cost. Nowadays grams of protein could be prepared using a large-scale cell-free system. Recently, the advantages on the expression of complicated, toxic and membrane proteins have shown the great potential of the CFPS systems. The rapid progress of this technology made us to believe that it will take an important place in biopharmaceutical industries undoubtedly.