Abstract: In this study,two- step method of silica gel chromatography was established to remove the defoamer in broth of antimicrobial lipopeptides for the first time.High- performance liquid chromatography( HPLC) was applied to detect the concentration of surfactin. Thin layer chromatography( TLC)- visible spectrophotometry was used to conduct quantitative analysis of triglyceride in eluent. Also,thin layer chromatography( TLC) was taken as the way to select the developing agent for triglyceride. The result showed,when 200 ~300 mesh silica gel was used as the chromatography support beads,the total loading amount of silica gel was 12.5mg / g. Under the condition of the mixture( petroleum ether∶ ethyl acetate∶ ethyl acid = 20 ∶ 2 ∶ 1,proportation of volume) as the fluid phase,flow rate of1 m L / min,97.97% of all the defoamer was obtained in the eluent. Secondly,when absolute ethyl alcohol was used as the fluid phase with a flow rate of 1m L / min,surfactin could be desorpted from the silica gel column,and its recovery reached 91.52%. The purification was greatly improved from 34.07% to 93.12%. This research provided experimental basement for the industrial production and application of antimicrobial lipopeptides.