Abstract: The optimum experimental conditions for the determination of L-tryptophan were investigated by fluorescence spectrophotometry using tetra-(4-Bocthreonine)-aminophenylporphyrin as the fluorescence spectral probe. Experiments indicated that the sensitivity of the system can be enhanced significantly by the addition of bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Under optimum experimental conditions, the decreased of the fluorescence intensity of the system was in proportion to the concentration of L-tryptophan in the concentration range of 0.10~8.80 μg/mL with a limit of detection of 0.034 μg/mL. The method has been used in the determination of samples with satisfactory results.