A reliable and cost-efficient protein synthesis system is the prerequisite for both structural and functional proteomic studies. The last decades saw the great technological improvement in the development of different protein synthesis systems. The cell-free protein synthesis system, especially Escherichia coli-based cell-free protein synthesis system, has emerged as one of the most robust protocols which can meet the growing demand of protein synthesis. E. coli-based cell-free protein synthesis system has become a more complete system for protein synthesis over the last two decades. Here, we review the main development and modifications of the E. coli-based cell-free protein synthesis system.