A reversed phase high performance liquid chromatography-fluorescence detection(RP-HPLC/FLD) method was developed for the determination of thiamine(T) and thiamine monophosphate(TMP),thiamine diphosphate(TDP) in human plasma，erythrocytes and urine.After deproteinization with perchloric acid and derivation with potassium ferricyanide,the sample was separated on a C18 column(250 mm×4.6 mm,5 μm i.d.) at 20 ℃.A gradient programme was carride out at flow rate of 0.8 mL/min using 0.2 mol/L KH2PO4( including 0.000 3 mol/L TBAH,pH 7.0 )-methyl alcohol and methyl alcohol-water as eluent.The excitation and emission wavelengths were set at 365 nm and 435 nm,respectively.The results showed that T,TMP and TDP could be completely separated within 18 min.The calibration curves were linear in the range of 0.5-20.0 μg/L for T and TMP,and 5.0-200.0 μg/L for TDP,with correlation coefficients of 0.99,0.98,0.99,respectively.The spiked recoveries of three compounds in plasma,erythrocytes and urine were in the range of 86.8%-110.2% with relative standard deviations(RSDs) of 2.5%-10.9%.The limits of detection(LODs) and limits of quantitation(LOQs) for T,TMP,TDP in plasma and erythrocytes were in the range of 0.03-0.35 μg/L,and 0.09-1.18 μg/L,respectively.The LODs and LOQs of T in urine were 1.58 μg/L and 5.26 μg/L,respectively.The developed method was sensitive and precise,and could meet the demands for clinical application.