The invention discloses a method for detecting isomers in irinotecan hydrochloride injection. An isocratic elution method is used for detection on a high performance liquid chromatograph, and a chromatographic column is a bonded chiral chromatographic column; a mobile phase is a mixture of methanol, absolute ethyl alcohol, normal hexane and diethylamine; the detection wavelength of an ultravioletdetector is 370nm; the flow velocity of the mobile phase is 1.7 mL/min to 1.9 mL/min; the column temperature is 30-40 DEG C; and the diluent is a mixture of phosphoric acid and absolute ethyl alcohol.The method can eliminate baseline fluctuation and interference of unknown impurities, achieves separation and determination of irinotecan hydrochloride injection isomer impurities, and has the advantages of high accuracy and strong specificity.