The invention discloses a preparation method of a lipopeptide biosurfactant. Bacillus subtilis G-34 is taken as a fermentation strain, and xylose is taken as a main carbon source to be fermented to prepare the biosurfactant; the registration collection number of the strain is CCTCC NO: M2014003, wherein the carbon source is xylose, a xylose mother liquor or an acidolysis solution of agricultural by-products; a fermented nitrogen source selects cheap biomass waste such as feather hydrolysis waste liquid and monosodium glutamate fermentation waste liquid; acetic acid is added to a fermentation medium to promote the strain fermentation so as to prepare the biosurfactant. The method disclosed by the invention efficiently utilizes cheap agricultural waste as a raw material to produce the lipopeptide biosurfactant so as to change the agricultural waste into valuables, so that the production cost of the lipopeptide biosurfactant is greatly lowered to provide technical support for industrial production of the lipopeptide biosurfactant.