A new spectrofluorimetric method was developed for the determination of trace amount of 5-hydroxytryptamine (5-HT) in human urine and serum samples. In the NaAc-HAc buffer solution of pH=5.80, 5-HT can react with formaldehyde-acetylacetone system to form a new compound which sends yellow green fluorescence at 533nm and the enhanced fluorescence intensity is in proportion to the concentration of 5-HT. Optimum conditions for the determination of 5-HT were also investigated. The dynamic range and detection limit for the determination of 5-HT are 5.35×10−7∼1.07×10−4 mol/L and 2.08×10−7 mol/L, respectively. The developed method is simple, practical and can be successfully applied to determination of 5-HT in human urine and serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the 5-HT - formaldehyde-acetylacetone system have been also discussed.