In view of the problems of high cost，long time and technical difficulty in the extraction and synthesis of messenger molecule （pp）pGpp，a new in vitro synthesis technology was established and optimized based on the existing in vitro enzymatic synthesis of （pp）pGpp.This method achieves the goals of high efficiency，convenience，safety and environmental protection，and low cost.The results showed that high purity （pp）pGpp can be obtained by reacting （pp）pGpp synthases：RelA，GppA and YvcI in 25 mmol/L Tris-HCl pH 9.0，15 mmol/L MgCl₂，100 mmol/L NaCl at 37 °C for 30 minutes，and further purified by anion exchange （pp）pGpp molecule.Compared with the traditional direct extraction from bacteria or plants and the existing in vitro enzymatic preparation process，this method has the advantages of simple operation，rapidity，low cost，and environmental friendliness，and can meet the needs of downstream biochemical analysis and structural biology experiments.More importantly，it provides a material basis for microbial signaling pathways and the development of new antibacterial drugs.