Cell-free synthetic biology has become a robust technology platform for synthesizing proteins and chemicals in recent years. Cell-free synthetic biology system activates biological machinery without the use of living cells, which opens new opportunities for the addition or synthesis of toxic substances at high concentrations in biological systems. Although it is generally accepted that an in vitro cell-free synthesis system has a high tolerance ability to toxic substances, there is a lack of relevant data to support this view. To explore particular tolerance ability, a range of different surfactants, lipids, materials, biofuels, and chemical drugs were selected for testing their effects in Escherichia coli-based cell-free protein synthesis system. The results showed the limit concentrations of different toxic substances. Moreover, the results demonstrated that the tolerance ability of a cell-free system is much higher than that of a cell system. This study further provides a rationale for the synthesis of toxic biopharmaceuticals, biochemicals, and biofuels by using cell-free systems.