A method for determination of tryptophan impurity M in amino acid injectionof Clinimix E.was established.HPLC was used to determine, octadecylsilane silica gel was used as filler, sodium dihydrogen phosphate buffer (pH=2.3)-acetonitrile (volume ratio 88.5:11.5) was used as mobile phase A, sodium dihydrogen phosphate buffer (pH=2.3)-acetonitrile (volume ratio 65:35) was used as mobile phase B, gradient elution was adopted, the flow rate was 0.7 mL·min-1, the detection wavelength was 220 nm, the column temperature was 40°C.The results showed that, there was good linear relationship between the mass concentration of tryptophan impurity M and the peak area method at the range of 0.2028∼4.0560μg·mL-1.The RSD values for the repeatability and accuracy of the method were 0 and 0.2%, resp.The method has good specificity, reproducibility and accuracy, and can be used for the determination of tryptophan impurity M content in Clinimix E.