The invention discloses a method used for preparing sulphoquinovosyl diglyceride (SQDG). The method comprises following steps: dried algae powder is dissolved and stirred in a extracting solution I for 4 to 5h under normal pressure at room temperature, a centrifugal machine is used for separating and collecting an organic phase I, the organic phase I is added into an extracting solution II, the centrifugal machine is used for separating and collecting an organic phase II, the organic phase II is delivered through a low-pressure chromatographic column for purification, an obtained product is subjected to vacuum concentration until weight is constant so as to obtain total glyceride; Q Sepharose column chromatography isolation and elution peaks are adopted for thin layer chromatography detection, an eluent is collected, and is subjected to vacuum concentration until weight is constant so as to obtain a SQDG crude product; hydrophobic chromatography separation and elution peaks are adopted for thin layer chromatography detection, and an eluent of SQDG is subjected to vacuum concentration until weight is constant so as to obtain a SQDG pure product; and the SQDG pure product is dissolved in water, and is subjected to ultrafiltration and freeze drying so as to obtain SQDG dry powder. The method used for preparing SQDG is simple; operation is safe; the extracting solutions are beneficial for increasing of SQDG yield; column chromatography and hydrophobic chromatography are combined to separate total glyceride, SQDG with relatively high purity can be obtained, and pollution of SQDG by other glycerides is avoided.