The vitamin B1 in rice was extracted by acid hydrolysis, enzymatic hydrolysis and oxidation. A C18 column was used for separation and elution. The mobile phase was methanol and sodium acetate (0.05 mol/L, pH=4.5, 35∶65). The vitamin B1 was detected by fluorescence detector quantificationally. The results showed that vitamin B1 had a good linear relationship in the concentration range of 0 ~ 0.20 μg/mL. The detection limit was 0.65 ng/mL. The recoveries of the three levels were above 95%. The relative standard deviations were 2.93% (n=6). There was no conspicuous difference between the result by this method and the result by the method described in GB/T 7628-2008.