The study was aimed at evaluation of the measurement uncertainty in the determination of 2，4-dichloropheNO_xyacetic acid(2，4-D) in water soluble fertilizers by high performance liquid chromatography. The sources of uncertainty in the testing process including sample weighing，volume measurement，the preparation of standard solution，the fitting of the standard curve，repeatability，recovery rate，and instrument precision were quantified and the expand uncertainty was calculated. The content of 2，4-D in a sample with the form of its expanded uncertainty was(391.0±29.4)μg/kg，k=2，at probability 95% level. The results showed that the uncertainty of this method mainly came from the preparation of standard solution，the fitting of standard curve and repeatability measurement.