A method of high performance liquid chromatography with fluorescence method was developed for determination of morphine and codeine. The liquid chromatographic experiment was carried out under the following conditions: the detector was a fluorescence with excitation wavelength at 285 nm and emission wavelength at 340 nm. An C18 column(250 mm×4.6 mm，5 μm) was used. The mobile phase was a mixture of methanol and 0.01 mol/L ammonium acetate（40∶60,v/v）. The flow rate was 1.0 mL/min. The result showed that a good linear relationship between mass concentration and corresponding peak area was the feeding in range of 0.5 ng to 0.02 μg. The lowest detection limit was 0.12 ng. The average recovery of sample for morphine and codeine was 91.1％～94.6％. The method is rapid, accurate, sensitive and suitable for the separation and determination of morphine and codeine in hot pot condiment.