The present invention provides a polymyxin B sulfate crystal and a preparation method thereof. Said method comprises the following steps of: adsorbing a polymyxin B fermentation liquid with a resin, and adding sulfuric acid or acidic ethanol for desorbing； concentrating to obtain a saturated solution of polymyxin B sulfate； and using an organic solvent to precipitate a crystal from the saturated solution, filtering and drying to obtain the polymyxin B sulfate crystal.
