A sample preparation method was developed to simulate the process of intracellular metabolites metabonomics analysis of Escherichia coli. The Escherichia coli cell was firstly quenched with cold sodium chloride solution ( 0. 85 %，precooled at -80℃ for 15 min).The quenched bacterial cell was treated by using the technique of vacuum freeze-drying and liquid nitrogen freezing combined with ultrasonic processing to increase cell membrane penetrability. Finally，a cold aqueous solution of methanol (MeOH:H2O, 1:1，V/ V, 4 ℃) was used as extraction solvent to extract metabolites. In the present research，flow cytometry and OD value recovery were performed to evaluate the degree of cell damage caused by quenching at single cell level and at integral level respectively. The tested results indicated that the degree of damage to cells caused by cold sodium chloride solution was less than 5%. The peak quantity and the total ion intensity detected by LC-TOF in low collision energy were used to evaluate extraction effects. Three different cell membrane penetrability modes and 4 kinds of extraction solvents were investigated and compared.The results showed that the technique of liquid nitrogen freezing combined with ultrasonic processing for cell membrane penetrability and a cold aqueous solution of methanol (MeOH/H2O，1:1，V/V, 4℃) for extraction of metabolites had the best extraction effect(peak quantity was greater than or equal to 105，and total ion intensity was in the range of 106-107).Therefore，in this work，the freeze drying，grinding with liquid nitrogen and ultrasonic extraction were combined to extract metabolites. In this way，it effectively promoted cell lysis and improved the efficiency of extraction. The result of synthetic analysis showed that the method proposed here could meet the requirements of the metabonomics analysis of Escherachaa coli.