A new method for separation and extraction of antibiotic lipopeptides produced by Bacillus amyloliquefaciens ES-2 with macroporous resin was carried out in this study. Among widely used twelve macroporous resins, X-5 resin had the highest adsorption capacity in static and dynamic tests, its non-polarity and configuration were suited for the lipopeptides (surfactin and fengycin). Dynamic adsorption and desorption experiments were performed using a column packed with X-5 resin to obtain optimal conditions, with 30 g/L resin concentration, 0.5 BV/h feed flow rate, 80% aqueous ethanol at 1 BV/h elute flow rate as eluant agent, the total content of lipopeptides in final product was 79.5% (surfactin content was 33.2% while fengycin content was 23.4%), which had been improved about 150% compared with that of lipopeptide in fermentation broth. Because of its low cost, high efficiency, procedural simplicity and scale up ease, the adsorption-desorption method could be more efficient for the preparative separation and extraction of antibiotic lipopeptide than the conventional acid precipitation method, which could provide scientific references for antibiotic lipopeptide production.