The invention relates to the field of biochemical analysis and specifically discloses a method for quickly detecting polypeptide. According to the invention, an ion exchange column is applied to an HPLC instrument platform and polypeptide in a to-be-tested sample can be quickly tested by taking a small amount of sample. The method comprises the following steps: preparing a to-be-tested sample solution from a sample in a fermenting or purifying process; performing gradient eluting with anion exchange resin as a filler of a chromatographic column, a phosphate buffer solution or Tris-HCl buffer solution as an eluant A, and a NaCl solution or KCl solution as an eluant B, thereby effectively separating polypeptide from impurities, such as impure protein and nucleic acid. The detecting method provided by the invention has the advantages of wide linear scope, repeatability, sampling recovery rate, lower limit of detection, simple operation and high sensitivity and can be used for quickly detecting polypeptide in the to-be-tested sample.