An HPLC method for determination of natamycin was established. A C18 column(4.6 mm×250 mm，5 μm) was  used with the phase of ammonium acetate-acetonitrile（760∶240）， at  the detection wavelength of  303 nm and the column temperature of 45 ℃.The calibration curves of natamycin were liner in the concentration rages of  0.04~0.3 mg/mL. The regression equation was y＝52 196 216x + 202 922， r＝1.000.The recovery was 99.73%  with  RSD 0.74%.The method was simple, accurate and reproducible.