The invention provides a method for detecting a toxoflavin residue in foodstuff based on multifunctional purifying column purification-high performance liquid chromatography. 80mL of acetonitrile and 20mL of water are used for dissolving a foodstuff sample, ultrasonic extraction is carried out, a multifunctional purifying column is used for purifying, 5.0mL of purifying liquid is used, N2 is used for blowing and drying, 1.0mL of water is used for metering the volume, and liquid for testing is obtained. Detection of the toxoflavin residue in the sample is realized, and the method has the advantages of good purifying effects, accurate quantification, and elimination of influences of solvent effect on chromatographic behaviors of toxoflavin. Gradient elution of a mobile phase is carried out by methanol and water, retention of toxoflavin in a reversed phase chromatography column, effective separation between toxoflavin and other interfering substances is realized during sample analysis, so that the purpose of accurate quantification is realized. In the sample purifying process, interferences of lipids, pigments, proteins and other substances are effectively removed from the sample, the purifying effect is good, and the quantitative analysis has good accuracy, high sensitivity, and good result reappearance; and the method is suitable for detection of the toxoflavin residue in a plurality of foodstuffs.